TAC1b mutation in Candida auris decreases manogepix susceptibility owing to increased CDR1 expression

Abstract

Candida auris is an emerging pathogenic fungus that is highly resistant to existing antifungal drugs. Manogepix is a novel antifungal agent that exerts antifungal activity by inhibiting glycosylphosphatidylinositol anchor biosynthesis. Although the mechanisms of resistance of Candida species to manogepix have been reported previously, those of C. auris are yet to be studied. To investigate the resistance mechanisms of C. auris, we exposed a clinical isolate (clade I) to manogepix in vitro and generated strains with reduced susceptibility to manogepix. A search for gain-of-function mutations that upregulate efflux pump expression confirmed the presence of the D865N amino acid mutation in TAC1b. We used the clustered regularly interspaced short palindromic repeats-Cas9 system to create a recovery strain (N865D) in which only this single nucleotide mutation was returned to the wild-type sequence. We generated a mutant strain by introducing only the D865N mutation into the parent strain and a different clade strain (clade III). The D865N mutant strains were clearly less susceptible to manogepix than the parental strains and exhibited high CDR1 expression. Moreover, we generated a strain deficient in CDR1 and confirmed that this strain had significantly increased susceptibility to manogepix. Thus, the present study demonstrated that the TAC1b mutation in C. auris upregulates CDR1 expression and decreases its susceptibility to manogepix.

Keywords: CDR1; CRISPR-Cas9; Candida auris; TAC1b; antifungal resistance; efflux pumps; manogepix.

Fig 1

Mutation in TAC1b reduced manogepix susceptibility in C. auris. The effect of D865N mutation in TAC1b on manogepix susceptibility was examined using a spot dilution assay in the wild-type and TAC1b^D865N mutants. Log-phase cells were serially diluted and spotted onto SC agar plates containing a gradient of manogepix. The plates were then incubated at 30°C for 48 h.

Fig 2

Mutation in TAC1b increased the expression level of CDR1 and activated the efflux pump. (A) Intracellular concentration of the fluorescent dye Nile red, a substrate of the efflux pump, was measured through flow cytometry. Nile red accumulation was reduced in the TAC1b D865N mutants. Median fluorescence intensity (MFI) was measured in three independent experiments. The mean ± standard error is shown. Statistical analysis was performed using two-tailed Student’s t-test. *P < 0.05, **P < 0.01. (B) Real-time qRT-PCR was performed to examine the expression of efflux pump-related genes. Gene expression level of NCPF8985 was defined as one in each assay and was normalized to that of ACT1. The expression levels of CDR1 were upregulated in TAC1b D865N mutants with decreased susceptibility to manogepix. The mean ± standard error of three independent experiments is shown.

Fig 3

Loss of Cdr1 or Tac1b restored manogepix susceptibility in the TAC1b mutant strain. The effect of deletion of CDR1 or TAC1b on manogepix susceptibility was examined using a spot dilution assay in the wild-type and TAC1b^D865N mutant. Log-phase cells were serially diluted and spotted onto SC agar plates containing a gradient of manogepix. The plates were then incubated at 30°C for 48 h.

Fig 4

Combination of efflux pump inhibitors restored manogepix susceptibility in TAC1b-mutant strains. The effect of clorgyline, an efflux pump inhibitor, in combination with manogepix was examined using a spot dilution assay in wild-type and TAC1b^D865N mutants. Log-phase cells were serially diluted and spotted onto SC agar plates containing the indicated concentrations of manogepix and clorgyline. The plates were then incubated at 30°C for 48 h.

Fig 5

Mutation in TAC1b reduced fluconazole susceptibility in C. auris. The effect of D865N mutation in TAC1b on manogepix susceptibility was examined using a spot dilution assay in the wild-type and TAC1b^D865N mutants. Log-phase cells were serially diluted and spotted onto SC agar plates containing 512-µg/mL fluconazole. The plates were then incubated at 30°C for 48 h.