The contribution of MRR2 mutations to fluconazole resistance and CDR1 expression in a collection of azole-resistant Candida albicans clinical isolates

Andrew T. Nishimoto1, Qing Zhang1, Brandon Hazlett1, Joachim Morschhäuser2, P. David Rogers1

1University of Tennessee Health Science Center, Memphis, TN
2Universität Würzburg, Würzburg, Germany

Background: Mutations in genes encoding zinc cluster transcription factors (ZCFs), such as TAC1, MRR1, and UPC2, play a key role in azole antifungal resistance in Candida albicans. Amino acid substitutions in the ZCF Mrr2 have recently been reported to influence fluconazole susceptibility through overexpression of the gene encoding the Cdr1 efflux pump. The purpose of this study is to determine the contributions to resistance of mutations in MRR2 among isolates within a collection of azole-resistant clinical C. albicans isolates.

Methods: MRR2 was sequenced in 52 C. albicans clinical isolates (42 fluconazole-resistant and 10 fluconazole-susceptible isolates) previously characterized for mutations in TAC1, MRR1, UPC2, ERG11, and ERG3, as well as expression CDR1, MDR1, and ERG11. Polymorphic MRR2 alleles found to occur uniquely among resistant isolates were introduced into reference strain SC5314 using the SAT1 flipper cassette. Susceptibility tests were performed according to the CLSI broth microdilution method and by E-test strip (Biomérieux) plating on RPMI-agar. Relative mRNA abundances of target genes were measured via qPCR.

Results: Mutations in MRR2 resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 amino acid substitutions (S466L, A468G, S469T, T470N) previously reported in the literature to influence fluconazole susceptibility. Four additional non-synonymous mutations were discovered uniquely in fluconazole-resistant isolates leading to R45Q, A459T, V486M, and V582L amino acid substitutions. When introduced into both MRR2 alleles of isolate SC5314, no change in fluconazole MIC or CDR1 expression was observed for any of the mutations in MRR2 found uniquely among the fluconazole-resistant isolates in this collection. However, artificial activation of MRR2 increased resistance to fluconazole as well as CDR1 expression. Moreover, in our hands, amino acid changes in Mrr2 reported previously to have the strongest effect on fluconazole susceptibility and CDR1 expression also did not cause increased CDR1 expression or fluconazole resistance relative to the parent strain. 

Conclusions: While all of the known mechanisms of fluconazole resistance are represented within this collection of clinical isolates and contribute to their resistance to fluconazole to different extents, no activating mutations in MRR2 that would alter expression of CDR1 or contribute to resistance were found in any of these isolates.