A Candidozyma ( Candida ) auris -optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of
Objectives:
Mutations in the Candidozyma ( Candida ) auris β-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 E pisomal P lasmid- I nduced C as9 (EPIC) gene-editing system capable of recyclable precision genome editing and demonstrate the contribution of FKS1 S639F mutation to echinocandin resistance.
Methods:
The 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 Fks1 S639F and Fks1 WT 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 β-glucan was assessed by staining with Calcofluor White (CFW) and Aniline Blue (AB).
Results:
The 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 FKS1 S639F mutation, resulted in a restoration of echinocandin sensitivity. Evaluation of cell wall composition showed isolates or strains harboring FKS1 S639F to contain significantly elevated β-glucan and chitin content relative to isogenic comparators.
Conclusions:
These 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.
Insights
A novel gene-editing system precisely modifies Candida auris FKS1, confirming the S639F mutation directly causes echinocandin resistance and alters cell wall composition. This advance aids in understanding and combating resistant fungal infections.
Area of Science:
- Mycology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Candida auris exhibits frequent echinocandin resistance linked to FKS1 gene mutations.
- The direct impact of specific FKS1 mutations, like S639F, on resistance is not fully characterized.
Purpose of the Study:
- To develop and utilize a novel CRISPR-Cas9 gene-editing system for Candida auris.
- To precisely introduce and assess the FKS1 S639F mutation's contribution to echinocandin resistance in C. auris.
Main Methods:
- Developed an optimized episomal plasmid-induced Cas9 (EPIC) gene-editing system for C. auris.
- Introduced FKS1 S639F and wildtype FKS1 into susceptible and resistant C. auris isolates.
- Assessed echinocandin susceptibility and cell wall composition (chitin, β-glucan).
Main Results:
- The EPIC system enabled precise FKS1 gene editing across multiple C. auris clades.
- Introduction of FKS1 S639F significantly increased echinocandin resistance.
- Correcting the FKS1 S639F mutation restored echinocandin susceptibility.
- Mutant strains showed elevated cell wall β-glucan and chitin content.
Conclusions:
- The EPIC system is effective for targeted genome editing in diverse C. auris clades.
- The FKS1 S639F mutation is a direct driver of echinocandin resistance in C. auris.
- Altered cell wall composition is associated with FKS1-mediated echinocandin resistance.
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