Mutations in TAC1B drive increased CDR1 and MDR1 expression and azole resistance in Candida auris
Objective: Candida auris has emerged as a fungal pathogen of particular concern owing in part to its propensity to exhibit antifungal resistance, especially to the commonly prescribed antifungal fluconazole. In this work we aimed to determine how mutations in the transcription factor gene TAC1B, which are common among resistant isolates and confer fluconazole resistance, exert this effect. Methods: Selected TAC1B mutations from clinical isolates were introduced into a susceptible isolate and reverted to the wild-type sequence in select clinical isolates using CRISPR Cas9 gene editing. Disruption mutants were likewise generated for select genes of interest. TAC1B mutants were subjected to transcriptional profiling by RNA-seq, and relative expression of specific genes of interest was determined by qRT-PCR. Antifungal susceptibilities were determined by modified CLSI broth microdilution. Results: TAC1B mutations leading to A640V, A657V, and F862_N866del conferred fluconazole resistance, as well as increased resistance to other triazoles, when introduced into a susceptible isolate. RNA-seq revealed that the ATP-Binding Cassette (ABC) transporter gene CDR1 as well as the Major Facilitator Superfamily (MFS) transporter gene MDR1 were both up-regulated by these TAC1B mutations. Disruption of CDR1 greatly abrogated resistance in strains with TAC1B mutations whereas disruption of MDR1 had little to no effect. However, disruption of both CDR1 and MDR1 resulted in an additional reduction in resistance as compared to disruption of either gene alone. Conclusion: TAC1B mutations leading to A640V, A657V, and F862_N866del all result in increased resistance to fluconazole and other triazole antifungals, and increased expression of both CDR1 and MDR1 in C. auris. CDR1 is the primary driver of resistance conferred by these TAC1B mutations. Overall design: Strains were constructed by CRISPR-Cas9 gene editing from a Candida auris Clade Ic clinical isolate (Kw2999) in order to correct the endogenous ERG11 mutation and then modify the endogenous TAC1B gene to either the wildtype sequence or to harbor one of the following amino acid substitutions: A640V, A657V, or F682_N866del. Each of the strains examined in this study were grown to mid-log phase in YPD at 35degC. For RNA-seq expression analysis, strain 1c (ERG11-WT/TAC1B-WT) was used as the control comparator for each of the TAC1B mutant strains.