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Updated: Jul 29, 2025
CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans
Published on: November 14, 2018
Evaluation of a Novel FKS1 R1354H Mutation Associated with Caspofungin Resistance in Candida auris Using the
Maiko Kiyohara1, Taiga Miyazaki1,2, Michiyo Okamoto3
1Department of Respiratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan.
Abstract:
Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistant Candida auris have been reported worldwide. Although hotspot mutations in FKS1 are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced the FKS1 gene of a caspofungin-resistant clinical isolate (clade I) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced into C. auris wild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to the FKS1 R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance in C. auris.
Insights
Multidrug-resistant Candida auris causes deadly infections. Researchers used CRISPR-Cas9 to confirm a novel FKS1 mutation (R1354H) drives echinocandin resistance, impacting treatment effectiveness.
Area of Science:
- Infectious Diseases
- Antimicrobial Resistance
- Molecular Biology
Background:
- Multidrug-resistant *Candida auris* outbreaks pose a significant global health threat.
- Echinocandin resistance in *C. auris* is linked to *FKS1* hotspot mutations, but their precise contribution is unclear.
- Understanding resistance mechanisms is crucial for effective antifungal treatment strategies.
Purpose of the Study:
- To investigate the specific role of a novel *FKS1* mutation (G4061A, R1354H) in caspofungin resistance in *Candida auris*.
- To assess the impact of this mutation on antifungal susceptibility and *in vivo* virulence.
- To demonstrate the utility of the CRISPR-Cas9 system in studying drug resistance mechanisms.
Main Methods:
- Sequencing of the *FKS1* gene in a caspofungin-resistant *C. auris* isolate.
- Utilizing CRISPR-Cas9 to generate specific revertant and mutant strains.
- Antifungal susceptibility testing (MIC determination) and *in vivo* mouse model of disseminated candidiasis.
Main Results:
- A novel *FKS1* mutation (G4061A, R1354H) was identified in a resistant isolate.
- Mutant strains with R1354H showed a 4- to 16-fold increase in caspofungin MIC.
- Reverted strains exhibited a 4-fold decrease in caspofungin MIC.
- In vivo efficacy of caspofungin correlated with the *FKS1* R1354H mutation and strain virulence, not solely in vitro MIC.
Conclusions:
- The *FKS1* R1354H mutation is a key driver of caspofungin resistance in *Candida auris*.
- CRISPR-Cas9 is a valuable tool for dissecting drug resistance mechanisms in *C. auris*.
- Treatment outcomes depend on both resistance mutations and intrinsic strain virulence.
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