Mutations in TAC1 and ERG11 are major drivers of triazole antifungal resistance in clinical isolates of Candida parapsilosis

Abstract

Objectives: The aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates.

Methods: Sequences of CpERG11 and CpTAC1 were determined for 35 resistant Candida parapsilosis clinical isolates. A plasmid-based CRISPR-Cas9 system was used to introduce mutations leading to amino acid substitution in CpTac1 and CpErg11. Triazole susceptibility was determined by broth microdilution and E-test. Differential expression of genes mediated by CpTAC1 mutation was determined by RNA sequencing, and relative expression of individual transporter genes was assessed with RT-qPCR.

Results: Six isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11^Y132F substitution. When introduced into susceptible isolates, this CpERG11 mutation led to a 4- to 8-fold increase in fluconazole minimum inhibitory concentrations (MIC; 0.125 μg/mL vs. 0.5 μg/mL, 0.125 μg/mL vs. 0.5 μg/mL, and 0.5 μg/mL vs. 4 μg/mL). When introduced into a susceptible isolate, the CpTAC1 mutation leading to the G650E substitution resulted in an 8-fold increase in fluconazole MIC (0.25 μg/mL vs. 2 μg/mL), whereas correction of this mutation in resistant isolates led to a 16-fold reduction in MIC (32 μg/mL vs. 2 μg/mL). CpCDR1, CpCDR1B, and CpCDR1C were overexpressed in the presence CpTac1^G650E. Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 μg/mL vs. 8 μg/mL).

Discussion: These results define the specific contribution made by the Y132F substitution in CpERG11 and demonstrate a role for activating mutations in CpTAC1 in triazole resistance in C. parapsilosis.

Keywords: Candida parapsilosis; ERG11; Fluconazole; Resistance; TAC1; Triazole.