In vitro evolution of caspofungin resistance in Candidozyma auris via FKS1 hotspot I mutations results in moderate fitness trade-offs but no reduction in virulence

Resistance to echinocandins in the recently emerged pathogen Candidozyma auris (previously known as Candida auris) poses significant clinical challenges. This study reports the in vitro evolution of a caspofungin-sensitive C. auris clade II isolate to acquire caspofungin resistance. We sequenced the whole genome of the parental strain and its drug-resistant progeny. The resistant isolates harboured mutations in the FKS1 gene altering a conserved residue (S639) also mutated in caspofungin-resistant clinical isolates; demonstrating that FKS1-S639 mutations are sufficient to confer resistance to caspofungin. Using these resistant strains, we examined cross-resistance & collateral sensitivity to other antifungal drugs and to cell wall stress, cell wall ultrastructure, biofilm formation, and virulence. As expected, both FKS1-S639P and FKS1-S639Y variants exhibited resistance not only to caspofungin but also to other echinocandins. Interestingly, these resistant strains showed moderate collateral sensitivity to certain azole antifungals and increased susceptibility to cell wall stress. While cell wall chitin content was slightly elevated in resistant isolates compared to sensitive ones, overall cell wall structure and phosphomannan levels remained unchanged. Biofilm formation capabilities were similar across all strains, and virulence in invertebrate models was unaffected by the FKS1 mutations. These findings indicate that while the evolution of caspofungin resistance through FKS1 mutations does incur fitness trade-offs, it does not substantially compromise the pathogenic potential of C. auris. This underscores the complexities of evolution of antifungal resistance mechanisms in this pathogen and the impact on its biology.