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.

Membrane Proteome-Wide Response to the Antifungal Drug Clotrimazole in Candida glabrata: Role of the Transcription Factor CgPdr1 and the Drug:H+ Antiporters CgTpo1_1 and CgTpo1_2

“…This is consistent with the characterization of several of these proteins as efflux pumps, despite a role in specific clotrimazole transport had only been described for CgQdr2 (31). The most up-regulated protein in this cluster is CgCdr1, widely characterized as an important ATP-binding cassette transporter.…”

“…Interestingly, at least one CgPdr1-binding site is found in the promoter regions of the first four genes, suggesting that the action of CgPdr1 in their expression may be direct. These results are consistent with the characterization of several of these proteins as efflux pumps and of their expression to be dependent on CgPdr1 in response to other chemical stress inducers (30,31,35,37). The expression changes here observed reinforce CgPdr1 as a major pleiotropic drug resistance mediator and highlight its role in mediating the expression of multidrug transporters in response to clotrimazole.…”

“…[ 3 H]-clotrimazole Accumulation Assays-3 H-clotrimazole transport assays were carried out as described before (31). The internal accumulation of clotrimazole was determined by calculating the ratio between the radiolabeled clotrimazole measured within the yeast cells and in the external medium (Intracellular/Extracellular).…”

Identification of Genomic Binding Sites for Candida glabrata Pdr1 Transcription Factor in Wild-Type and ρ 0 Cells

“…This is similar to the situation in S. cerevisiae, although there are two unique C. glabrata targets, YBT1 and QDR2. QDR2 has been observed by another group to be under the transcriptional control of Pdr1 (28). These data coupled with our ChIP-seq analysis provide arguably the strongest case for an MFS protein-encoding gene regulated by Pdr1.…”

Resistance reversal induced by a combination of fluconazole and tacrolimus (FK506) in Candida glabrata

“…Up-to-date research has highlighted the important role that complex resistance gene overexpression interactions play in FLC resistance in C. glabrata, and has demonstrated that overexpression of ABC membrane transporter genes is a pivotal FLC resistance mechanism in C. glabrata clinical isolates (Abbes et al, 2013). Although the MFS transporter genes FLR1 and QDR2 in C. glabrata are known to be involved in azole efflux, the expression of FLR1 and QDR2 in C. glabrata was not found to confer resistance to FLC (Chen et al, 2007;Costa et al, 2013). Therefore, the investigation of FLC resistance gene expression did not involve the MFS transporter genes in this work.…”