Precise editing using CRISPR-Cas9 to explore the contribution of clinically-derived mutations to antifungal resistance in the pathogenic yeast Candida parapsilosis

Abstract

Introduction Candida parapsilosis is both a commensal/saprophytic yeast of the human skin and an opportunistic pathogen which can be responsible for life-threatening infections. The increasing reports of clonal outbreaks involving azole-resistant C. parapsilosis in the clinical setting is worrisome and urges for a better understanding of antifungal resistance in this species. Previous studies have identified mutations in key genes which can explain acquired fluconazole resistance. Reverse genetics approaches are now warranted to confirm their involvement and to determine whether they can affect other clinically-licensed antifungals. Here, we used a CRISPR-Cas9 technique to study the relative contributions of clinically-derived mutations to antifungal resistance and provide answers to these questions. Materials and Methods Six clinically-derived mutations were selected (ERG11Y132F, ERG11K143R,ERG11R398I, TAC1G650E, MRR1G583R, ERG3G111R) to be engineered in two C. parapsilosis fluconazole-susceptible backgrounds (ATCC22019, STZ5) using a previously described CRISPR-Cas9 method. In vitro susceptibility of the transformants to fluconazole, voriconazole, posaconazole, isavuconazole and micafungin was determined by Etest®. Results/Discussion The impact on fluconazole susceptibility was highly variable depending on the residue/gene involved, but roughly similar between the two genetic backgrounds. All but two(ERG11R398I, ERG3G111R) conferred fluconazole resistance, though the highest MIC increase was observed for MRR1G583R (≥650 fold). As expected in a diploid species, we noted an impact of allelic dosage. Some kind of cross-resistance to the other azoles was noted from some mutations, although the impact was lower for posaconazole and isavuconazole, except for MRR1G583R which led to multi-azole resistance. Finally, ERG3G111R increased tolerance to both azoles and echinocandins.

Most azole antifungal resistance mutations in the drug target provide cross-resistance and carry no intrinsic fitness cost

“…We therefore think that our assays are conservative. Furthermore, a study in Candida parapsilosis also noted the absence of a cost for a resistance mutation in ERG11 in a Galleria mellonella model of infection 63 .…”