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

The aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates.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.Six isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11Y132F 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 CpTac1G650E. Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 μg/mL vs. 8 μg/mL).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.