Mutations inTAC1BdriveCDR1andMDR1expression and azole resistance inC. auris
Abstract
Objective
Candida aurishas 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 geneTAC1B, which are common among resistant isolates and confer fluconazole resistance, exert this effect.
Methods
SelectedTAC1Bmutations 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.TAC1Bmutants 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
TAC1Bmutations 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 geneCDR1as well as the Major Facilitator Superfamily (MFS) transporter geneMDR1were both upregulated by theseTAC1Bmutations. Disruption ofCDR1greatly abrogated resistance in strains withTAC1Bmutations whereas disruption ofMDR1had little to no effect. However, disruption of bothCDR1andMDR1resulted in an additional reduction in resistance as compared to disruption of either gene alone.
Conclusion
TAC1Bmutations leading to A640V, A657V, and F862_N866del all result in increased resistance to fluconazole and other triazole antifungals, and increased expression of bothCDR1andMDR1inC. auris.CDR1is the primary driver of resistance conferred by theseTAC1Bmutations.
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