Biological Functions Of IPF7817 In Candida Albicans

CaIPF7817,a functionally unknown gene in Candida albicans,was suggested to be involved in the redox system and drug resistance previously,but its exact role is unknown.In this study,biological functions of IPF7817 were investigated through the generation of ipf7817 null mutant.Firstly,ipf7817 null mutant was generated with the Ura-blaster method,and verificated through Southern hybridization analysis. Secondly,the effects of IPF7817 on the redox homeostasis were investigated.After the deletion of IPF7817,intracellulaer levels of reactive oxygen species(ROS)were significantly increased;mitochondrial membrane potential,a direct indicator of mitochondrial function,was elevated;some important redox-related genes,including GLR1,SOD2,and TRR1,were up-regulated;and the intracellular GSH/GSSG ratio was raised.These changes indicated that IPF7817 played important roles in the regulation of redox homeostasis in C.albicans.Thirdly,the effects of IPF7817 on drug resistance were studied.After the deletion of IPF7817,no apparent changes were observed on drug sensitivity and Rhodamine 6G(R6G)effiux,while the overexpression of multidrug resistance gene MDR1 was found in the ipf7817 mutant strains by the real-time RT-PCR analysis.Furthermore,fluconazole(6μg/mL and 24μg/mL)inducing drug resistance experiment was carried out on the mutant strain JH2 and the parent strain RM1000.Minimal inhibitory concentration(MIC)results showed that the emergence of drug resistance in the ipf7817 mutant strains was earlier than the parental strain,and the resistant strains were also resistant to itraconazole, ketoconazole.No cross resistance was observed to terbinafine and amphotericin B for the fluconazole resistant strains.Spot assay and Filter disk assay results confirmed the MIC results.Compared with the induced parental strains RM1000_F647and RM1000_F24-48,the ERG11(the azole target gene)expression was increased in the induced mutant resistant strains JH2_F6-47and JH2_F24-48.For the resistant parental strain induced by fluconazole of 6μg/mL,the drug susceptibility phenotype could be reverted and the MIC could reach 0.5μg/mL,while the induced resistant ipf7817 mutant strain remained resistant over 40 days in fluconazole-free medium.Additionally,the effects of IPF7817 on hyphae and biofilm formation were also investigated and no obvious differences were found after the deletion of IPF7817.Generally speaking,IPF7817 was involved in the regulation of redox homeostasis and the emergence of drug resistance in C.albicans.