Relative contributions of the ERG11VF125AL and MRR1AN647T mutations to fluconazole resistance in Clade III Candidozyma (Candida) auris clinical isolates

ObjectivesCandidozyma (Candida) auris is an emerging fungal pathogen of global concern that often exhibits multi-drug resistance. Over 90% of isolates are resistant to fluconazole. Of the six described clades of C. auris, Clade III has been found to be nearly universally fluconazole resistant and almost every Clade III isolate described carries a mutation in the gene encoding the fluconazole target sterol demethylase (ERG11) leading to a VF125AL substitution and a mutation leading to a N647T substitution in the gene encoding Mrr1a, a transcriptional regulator of the Mdr1 transporter. Both mutations have been shown to contribute to fluconazole resistance in C. auris. MethodsIn the present study we introduced the Clade III MRR1A mutation into a Clade I background using CRISPR-Cas9 gene editing. In two Clade III clinical isolates we corrected the native MRR1A and ERG11 mutations to their wild-type sequences as well as disrupted MDR1. Triazole susceptibilities and MDR1 gene expression were measured in all strains. ResultsIntroduction of the N647T substitution in a Clade I background confers a modest reduction in fluconazole and voriconazole susceptibility. Similarly, correction of MRR1A or disruption of MDR1 in each Clade III background resulted in a one-dilution decrease in fluconazole and voriconazole MIC while the ERG11 correction resulted in a three-dilution decrease in fluconazole and voriconazole MIC. ConclusionsOur findings show that while the MRR1A mutation makes a modest contribution, the ERG11 mutation is responsible for most of the fluconazole resistance observed in Clade III isolates. We also show that while these mutations likewise affect voriconazole susceptibility, they have no effect on susceptibility to itraconazole, isavuconazole, or posaconazole suggesting the potential therapeutic utility of these antifungals for infections due to Clade III isolates of C. auris.