Candida glabrata drug:H+ antiporter CgQdr2 confers imidazole drug resistance, being activated by transcription factor CgPdr1

Abstract

The widespread emergence of antifungal drug resistance poses a severe clinical problem. Though predicted to play a role in this phenomenon, the drug:H(+) antiporters (DHA) of the major facilitator superfamily have largely escaped characterization in pathogenic yeasts. This work describes the first DHA from the pathogenic yeast Candida glabrata reported to be involved in antifungal drug resistance, the C. glabrata QDR2 (CgQDR2) gene (ORF CAGL0G08624g). The expression of CgQDR2 in C. glabrata was found to confer resistance to the antifungal drugs miconazole, tioconazole, clotrimazole, and ketoconazole. By use of a green fluorescent protein (GFP) fusion, the CgQdr2 protein was found to be targeted to the plasma membrane in C. glabrata. In agreement with these observations, CgQDR2 expression was found to decrease the intracellular accumulation of radiolabeled clotrimazole in C. glabrata and to play a role in the extrusion of this antifungal from preloaded cells. Interestingly, the functional heterologous expression of CgQDR2 in the model yeast Saccharomyces cerevisiae further confirmed the role of this gene as a multidrug resistance determinant: its expression was able to complement the susceptibility phenotype exhibited by its S. cerevisiae homologue, QDR2, in the presence of imidazoles and of the antimalarial and antiarrhythmic drug quinidine. In contrast to the findings reported for Qdr2, CgQdr2 expression does not contribute to the ability of yeast to grow under K(+)-limiting conditions. Interestingly, CgQDR2 transcript levels were seen to be upregulated in C. glabrata cells challenged with clotrimazole or quinidine. This upregulation was found to depend directly on the transcription factor CgPdr1, the major regulator of multidrug resistance in this pathogenic yeast, which has also been found to be a determinant of quinidine and clotrimazole resistance in C. glabrata.

Fig 1

Fluorescence of exponential-phase C. glabrata L5U1 (A) and S. cerevisiae BY4741 (B) cells harboring either the cloning vector pGREG576 (control) or the pGREG576_MTII_CgQDR2 or pGREG576_CgQDR2 plasmid (CgQdr2_GFP) after 5 h of copper- or galactose-induced recombinant protein production, respectively. The results indicate that the CgQdr2-GFP fusion protein localizes to the plasma membranes of both S. cerevisiae and C. glabrata cells.

Fig 2

(A) Comparison by spot assays of the susceptibilities of C. glabrata strains KUE100 and KUE100_Δcgqdr2 to miconazole, clotrimazole, ketoconazole, fluconazole, itraconazole, and quinidine, at the concentrations indicated, in BM agar plates. (B) Comparison by spot assays of the susceptibilities of C. glabrata strain L5U1, harboring the pGREG576 cloning vector (v) or the pGREG576_CgQDR2 plasmid, to miconazole, clotrimazole, ketoconazole, and quinidine, at the indicated concentrations, in BM agar plates. The inocula were prepared as described in Materials and Methods. The cell suspensions used to prepare the spots were 1:5 (b) and 1:25 (c) dilutions of the cell suspension used for column a. The images displayed are representative of at least three independent experiments.

Fig 3

(A) Comparison by spot assays of the susceptibilities of S. cerevisiae strains BY4741 and BY4741_Δqdr2, harboring the cloning vector pGREG576 (v) or the CgQDR2 expression plasmid pGREG576_CgQDR2, to miconazole, tioconazole, and quinidine, at the indicated concentrations, in MM4 agar plates. The cell suspensions used to prepare the spots were 1:5 (b) and 1:25 (c) dilutions of the cell suspension used for column a. (B) Comparison of the susceptibilities of S. cerevisiae strains BY4741 (□, ◆) and BY4741_Δqdr2 (○, ▲), harboring the cloning vector pGREG576 (◆, ▲) or the CgQDR2 expression plasmid pGREG576_CgQDR2 (□, ○), under control conditions (A) and under stress induced by 7 mg/liter miconazole (B), 5 mg/liter tioconazole (C), or 4.5 mg/liter quinidine (D) in MM4 liquid medium. The inocula were prepared as described in Materials and Methods. Growth curves and spot assay images are representative of at least three independent experiments.

Fig 4

(A) Time course accumulation ratio of [³H]clotrimazole in nonadapted cells of KUE100 (filled diamonds) or KUE100_Δcgqdr2 (filled squares) strains during cultivation in liquid BM in the presence of 30 mg/liter unlabeled clotrimazole. (B) Time course efflux ratio of [³H]clotrimazole in preloaded KUE100 (diamonds) or KUE100_Δcgqdr2 (squares) cells, upon a glucose pulse, in the presence (open symbols) or absence (filled symbols) of the protonophore CCCP, given after 30 min of passive accumulation of the radiolabeled drug. The accumulation ratio values are averages for at least three independent experiments. Error bars represent standard deviations.

Fig 5

(A) Comparison by spot assays of the susceptibilities of C. glabrata strains 66032 and 66032_Δcgpdr1 to clotrimazole and quinidine, at the indicated concentrations, in BM agar plates. The inocula were prepared as described in Materials and Methods. The cell suspensions used to prepare the spots were 1:5 (b) and 1:25 (c) dilutions of the cell suspension used in column a. The images displayed are representative of at least three independent experiments. (B) Comparison of the differences in CgQDR2 transcript levels in C. glabrata 66032 wild-type (filled bars) and Δcgpdr1 (shaded bars) cells before (control) and after 1 h of exposure to stress induced by quinidine or clotrimazole at the indicated concentrations. Transcript levels were determined by quantitative RT-PCR, as described in Materials and Methods, and are expressed as CgQDR2/CgACT1 mRNA ratios. The value reported for wild-type cells under control conditions was considered equal to 1. The values shown are averages for at least three independent experiments. Error bars represent standard deviations.

Fig 6

(A) CgQDR2 promoter analysis based on a CgQDR2 promoter-lacZ fusion. The changes in β-galactosidase activity, expressed from wild-type, ΔPDRE1, and ΔPDRE2 CgQDR2 promoter-lacZ fusions, in wild-type L5U1 cells under control conditions (filled bars) or upon exposure for 2 h (shaded bars) or 4 h (open bars) to 30 mg/liter clotrimazole were compared. The values obtained are averages for at least three independent experiments. Error bars represent standard deviations. (B) Schematic representation of the occurrence of predicted CgPdr1 binding sites (GCCATCATT and GCCGATAGA [34]) in the CgQDR2 promoter region, as analyzed with the YEASTRACT database tools (44).