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.

Opportunistic Candida species often have to cope with inhibitory concentrations of acetic acid, in the acidic environment of the vaginal mucosa. Given that the ability of these yeast species to tolerate stress induced by weak acids and antifungal drugs appears to be a key factor in their persistence and virulence, it is crucial to understand the underlying mechanisms. In this study, the drug:H+ antiporter CgAqr1 (ORF CAGL0J09944g), from Candida glabrata, was identified as a determinant of resistance to acetic acid, and also to the antifungal agents flucytosine and, less significantly, clotrimazole. These antifungals were found to act synergistically with acetic acid against this pathogen. The action of CgAqr1 in this phenomenon was analyzed. Using a green fluorescent protein fusion, CgAqr1 was found to localize to the plasma membrane and to membrane vesicles when expressed in C. glabrata or, heterologously, in Saccharomyces cerevisiae. Given its ability to complement the susceptibility phenotype of its S. cerevisiae homolog, ScAqr1, CgAqr1 was proposed to play a similar role in mediating the extrusion of chemical compounds. Significantly, the expression of this gene was found to reduce the intracellular accumulation of 3H-flucytosine and, to a moderate extent, of 3H-clotrimazole, consistent with a direct role in antifungal drug efflux. Interestingly, no effect of CgAQR1 deletion could be found on the intracellular accumulation of 14C-acetic acid, suggesting that its role in acetic acid resistance may be indirect, presumably through the transport of a still unidentified physiological substrate. Although neither of the tested chemicals induces changes in CgAQR1 expression, pre-exposure to flucytosine or clotrimazole was found to make C. glabrata cells more sensitive to acetic acid stress. Results from this study show that CgAqr1 is an antifungal drug resistance determinant and raise the hypothesis that it may play a role in C. glabrata persistent colonization and multidrug resistance.

Azoles are widely used antifungal drugs. This family of compounds includes triazoles, mostly used in the treatment of systemic infections, and imidazoles, such as clotrimazole, often used in the case of superficial infections. Candida glabrata is the second most common cause of candidemia worldwide and presents higher levels of intrinsic azole resistance when compared with Candida albicans, thus being an interesting subject for the study of azole resistance mechanisms in fungal pathogens.Since resistance often relies on the action of membrane transporters, including drug efflux pumps from the ATPbinding cassette family or from the Drug:H ؉ antiporter (DHA) 1 family, an iTRAQ-based membrane proteomics analysis was performed to identify all the membraneassociated proteins whose abundance changes in C. glabrata cells exposed to the azole drug clotrimazole. Proteins found to have significant expression changes in this context were clustered into functional groups, namely: glucose metabolism, oxidative phosphorylation, mitochondrial import, ribosome components and translation machinery, lipid metabolism, multidrug resistance transporters, cell wall assembly, and stress response, comprising a total of 37 proteins. Among these, the DHA transporter CgTpo1_2 (ORF CAGL0E03674g) was identified as overexpressed in the C. glabrata membrane in response to clotrimazole. Functional characterization of this putative drug:H ؉ antiporter, and of its homolog CgTpo1_1 (ORF CAGL0G03927g), allowed the identification of these proteins as localized to the plasma membrane and conferring azole drug resistance in this fungal pathogen by actively extruding the drug to the external medium. The cell wall protein CgGas1 was also shown to confer azole drug resistance through cell wall remodeling.