Genomewide Elucidation of Drug Resistance Mechanisms for Systemically Used Antifungal Drugs Amphotericin B, Caspofungin, and Voriconazole in the Budding Yeast

Abstract

There are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up- or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.

Keywords: amphotericin B; antifungal agents; caspofungin; drug resistance; genomics; multidrug resistance; voriconazole.

FIG 1

Spotting assay for AmB, CSP, and VOR resistance genes. (A) Spotting assay of wild-type cells growing on agar plates with the indicated concentrations of antifungal drugs. The cells were serially diluted, and a 5-μl aliquot was dropped for each spot. (B) Wild-type cells overexpressing PDR16 and PMP3, RMD9 and SWH1, or MRS3 and TRI1 were grown on plates containing a gradient of AmB, CSP, and VOR, respectively. Wild-type cells with an empty plasmid were used as controls (p-only). (C) Spotting assay for deletion mutants. The plates were incubated at 30°C for 3 days after inoculation and photographed.

FIG 2

Real-time qPCR analyses for resistance genes. Wild-type cells were incubated with a sublethal dose of AmB (0.2 μg/ml) (A), VOR (75 μg/ml) (B), and CSP (0.01 μg/ml) (C) for 2 h and analyzed for their transcript levels. The ACT1 gene used as an internal control. The significance of the differences was calculated by Student’s t test (P < 0.05). Each gene was analyzed in two biological samples with triplicate readings. The error bars indicate SD.

FIG 3

Plasma membrane potential measurements. (A) Relative membrane potentials of the deletion mutants. BY4741 wild-type cells were used as the control. (B) Membrane potentials of wild-type cells overexpressing PDR16 and PMP3 genes. Cells carrying empty plasmids (p426) were used as the control. (C) Membrane potentials of wild-type cells overexpressing RMD9 and SWH1 genes. Cells carrying empty plasmids (pAG425) were used as the control. (D) Membrane potentials of wild-type cells overexpressing MRS3 and TRI1 genes. Cells carrying empty plasmids (pAG425) were used as the control.

FIG 4

Intracellular pH values of the mutants. The error bars indicate SD.

FIG 5

Test of C. albicans orthologous genes. Candida genes were cloned into yeast expression vectors and expressed in S. cerevisiae. (A) Cells were grown on an AmB gradient between 0 μg/ml and 1.5 μg/ml. (B) Cells were grown on a VOR gradient between 0 μg/ml and 150 μg/ml.