Exploration of novel mechanisms of azole resistance in <i>Candida auris</i>

<i>Candida auris</i> is a pathogenic yeast of particular concern because of its ability to cause nosocomial outbreaks of invasive candidiasis (IC) and to develop resistance to all current antifungal drug classes. Most <i>C. auris</i> clinical isolates are resistant to fluconazole, an azole drug that is used for the treatment of IC. Azole resistance may arise from diverse mechanisms, such as mutations of the target gene (<i>ERG11</i>) or upregulation of efflux pumps via gain of function mutations of the transcription factors <i>TAC1</i> and/or <i>MRR1</i>. To explore novel mechanisms of azole resistance in <i>C. auris</i>, we applied an <i>in vitro</i> evolutionary protocol to induce azole resistance in a <i>TAC1A</i>/<i>TAC1B</i>/<i>MRR1</i> triple-deletion strain. Azole-resistant isolates without <i>ERG11</i> mutations were further analyzed. In addition to a whole chromosome aneuploidy of chromosome 5, amino acid substitutions were recovered in the transcription factor Upc2 (N592S, L499F), the ubiquitin ligase complex consisting of Ubr2 (P708T, H1275P) and Mub1 (Y765*), and the mitochondrial protein Mrs7 (D293H). Genetic introduction of these mutations in an azole-susceptible wild-type <i>C. auris</i> isolate of clade IV resulted in significantly decreased azole susceptibility. Real-time reverse transcription PCR analyses were performed to assess the impact of these mutations on the expression of genes involved in azole resistance, such as <i>ERG11</i>, the efflux pumps <i>CDR1</i> and <i>MDR1</i> or the transcription factor <i>RPN4</i>. In conclusion, this work provides further insights in the complex and multiple pathways of azole resistance of <i>C. auris</i>. Further analyses would be warranted to assess their respective role in azole resistance of clinical isolates.