An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway

ABSTRACT The World Health Organization recently published the first list of priority fungal pathogens highlighting multiple Candida species, including Candida glabrata, Candida albicans, and Candida auris. However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes, NatMX and HphMX, limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit for C. glabrata, C. auris, and C. albicans to include KanMX and BleMX when coupled with an in vitro assembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes for Candida, we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion of ERG3 in C. glabrata, C. auris, and C. albicans prototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that an ERG5 deletion in C. glabrata is azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when both ERG3 and ERG5 are deleted in C. glabrata, which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates. IMPORTANCE The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis. The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.