A Candidozyma (Candida) auris-optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of the S639F encoding FKS1 mutation

Abstract

ObjectivesMutations in the Candidozyma (Candida) auris {beta}-glucan synthase gene (FKS1) altering S639 are frequently associated with clinical echinocandin resistance. However, the direct impact of these mutations remains uncharacterized. We have developed a novel C. auris-optimized Episomal Plasmid-Induced Cas9 (EPIC) gene-editing system capable of recyclable precision genome editing and demonstrate the contribution of FKS1S639F mutation to echinocandin resistance. MethodsThe EPIC gene-editing system was generated for optimized use in C. auris, and ADE2 modification was evaluated in 5 C. auris clades. Mutations leading to Fks1S639F and Fks1WT were placed into echinocandin-susceptible and echinocandin-resistant isolates from Clade-III and -I, respectively, using the EPIC system. Echinocandin susceptibility was determined by CLSI broth microdilution, and cell wall abundance of chitin and {beta}-glucan was assessed by staining with Calcofluor White (CFW) and Aniline Blue (AB). ResultsThe EPIC system was capable of targeted ADE2 editing in C. auris isolates from 5 genetic clades and shown to be precise by confirmatory sequencing of 50 transformants. A single nucleotide change in FKS1 resulting in either the S639F substitution or a silent synonymous mutation was introduced in an echinocandin-susceptible Clade-III isolate. Precision FKS1 editing by the EPIC system was confirmed by whole genome sequencing. Subsequent susceptibility testing demonstrated introduction of the FKS1 S639F mutation to increase resistance to echinocandins. Moreover, introduction of a wildtype Fks1 sequence to an echinocandin-resistant Clade-I clinical isolate, correcting the FKS1S639F mutation, resulted in a restoration of echinocandin sensitivity. Evaluation of cell wall composition showed isolates or strains harboring FKS1S639F to contain significantly elevated {beta}-glucan and chitin content relative to isogenic comparators. ConclusionsThese data demonstrate the potential of our EPIC system in its ability to introduce single nucleotide substitutions in multiple C. auris clade backgrounds while revealing the direct impact of the S639F encoding FKS1 mutation on echinocandin resistance.