Candida glabrata drug:H+ antiporter CgTpo3 (ORF CAGL0I10384g): role in azole drug resistance and polyamine homeostasis

The ability of opportunistic pathogenic Candida species to persist and invade specific niches in the human host depends on their resistance to natural growth inhibitors and antifungal therapy. This work describes the role of the Candida glabrata drug:H+ antiporter CgTpo3 (ORF CAGL0I10384g) in this context. Deletion and cloning of CgTPO3 was achieved using molecular biology tools. C. glabrata strain susceptibility was assayed based on growth in liquid and solid media and through MIC determination. Radiolabelled compound accumulation or HPLC were used for the assessment of the role of CgTpo3 as a drug or polyamine transporter. Quantitative RT-PCR was used for expression analysis. CgTpo3 was found to confer resistance to azole drugs in C. glabrata. This protein was found to be localized to the plasma membrane and to decrease the intracellular accumulation of [H-3]clotrimazole, playing a direct role in its extrusion from pre-loaded C. glabrata cells. CgTPO3 was further found to confer resistance to spermine, complementing the susceptibility phenotypes exhibited by the deletion of its Saccharomyces cerevisiae homologue, TPO3. In spermine-stressed C. glabrata cells, CgTPO3 is transcriptionally activated in a CgPdr1-dependent manner, contributing to a decrease in the intracellular concentration of this polyamine. Clotrimazole exposure was found to lead to the intracellular accumulation of spermine, and pre-exposure to this polyamine was found consistently to lead to increased clotrimazole resistance. Altogether, these results point to a significant role for CgTpo3 in azole drug resistance and in the tolerance to high polyamine concentrations, such as those found in the urogenital tract.