Membrane Proteomics Analysis of the Candida glabrata Response to 5-Flucytosine: Unveiling the Role and Regulation of the Drug Efflux Transporters CgFlr1 and CgFlr2

Abstract

Resistance to 5-flucytosine (5-FC), used as an antifungal drug in combination therapy, compromises its therapeutic action. In this work, the response of the human pathogen <i>Candida glabrata</i> to 5-FC was evaluated at the membrane proteome level, using an iTRAQ-based approach. A total of 32 proteins were found to display significant expression changes in the membrane fraction of cells upon exposure to 5-FC, 50% of which under the control of CgPdr1, the major regulator of azole drug resistance. These proteins cluster into functional groups associated to cell wall assembly, lipid metabolism, amino acid/nucleotide metabolism, ribosome components and translation machinery, mitochondrial function, glucose metabolism, and multidrug resistance transport. Given the obtained indications, the function of the drug:H+ antiporters CgFlr1 (ORF <i>CAGL0H06017g</i>) and CgFlr2 (ORF <i>CAGL0H06039g</i>) was evaluated. The expression of both proteins, localized to the plasma membrane, was found to confer flucytosine resistance. CgFlr2 further confers azole drug resistance. The deletion of <i>CgFLR1</i> or <i>CgFLR2</i> was seen to increase the intracellular accumulation of 5-FC, or 5-FC and clotrimazole, suggesting that these transporters play direct roles in drug extrusion. The expression of <i>CgFLR1</i> and <i>CgFLR2</i> was found to be controlled by the transcription factors CgPdr1 and CgYap1, major regulator of oxidative stress resistance.