Research On The Relationship Between Missense Mutations In Mrr2p And Fluconazole Resistance To Candida Albicans

Object To investigate the correlation of missense mutation in Mrr2 p with fluconazole resistance to Candida albicans.Methods 1.Candida albicans isolates were collected from several hospitals in Shanghai.The minimum inhibitory concentrations(MICs)of fluconazole were evaluated using the broth microdilution reference method according to the guidelines M27-A3 of the Clinical and Laboratory Standards Institute(CLSI).2.Quantitative real-time reverse transcription PCR(RT-PCR)was performed to analyze the expression of CDR1 and CDR2 in clinical fluconazole-resistant and fluconazole sensitive-dose-dependent isolates,while the fluconazole-susceptible isolates were regarded as calibrator.3.To discover the potential gain-of-function(GOF)mutations in Mrr2 p,MRR2 genes were amplified and sequenced in fluconazole-susceptible isolates and C.albicans isolates which overexpressed CDR1.Meanwhile,TAC1 genes were sequenced in C.albicans isolates which overexpressed CDR1 or CDR2 to find the GOF mutations which have been reported.4.Strains expressing the mutated MRR2 alleles were generated using gene knockout technology and site-directed mutagenesis.Then fluconazole susceptibility testing and RT-PCR were performed to analyze the relationship between the mutations in MRR2 and fluconazole resistance.5.MLST analysis was conducted to investigate the epidemiological relationship among C.albicans isolates containing MRR2 missense mutations.Results 1.There were 20 fluconazole-resistant or sensitive-dose-dependent isolates were identified among 29 C.albicans isolates,while the remaining 9 isolates were fluconazole-susceptible.2.Of the 20 fluconazole-resistant C.albicans,12 isolates overexpressed CDR1,16 isolates overexpressed CDR2,while 17 isolates overexpressed CDR1 or CDR2.3.Among the clinical C.albicans isolates overexpressing CDR1,three(C7,C9,C15)contained 11 identical missense mutations,6(H358N,E439 K,S466L,A468 G,S469T and T470N)of which occurred only in fluconazole-resistant or sensitive-dose-dependent isolates.4.Compared with SN152,the strains expressing the mutated MRR2 alleles showed different levels of increase in MIC and overexpression in CDR1,especially the mutated MRR2 alleles from clinical isolate C9 contributed to an almost six-fold increase in CDR1 expression and an eight-fold increase in fluconazole resistance.5.Three clinical isolates containing MRR2 missense mutations are closely related in terms of molecular epidemiology.Conclusion Missense mutations in Mrr2 p are assuredly contributing fluconazole resistance to clinical C.albicans isolates.What’s more,the mechanisms of fluconazole resistance are relevant with CDR1 overexpression.The MIC of fluconazole and CDR1 overexpression were much more increased in strain containing all above MRR2 missense mutations.Missense mutations in Mrr2 p are not common in clinical C.albicans isolates.