A yeast cell-based system for screening Candida glabrata multidrug resistance reversal agents and selection of loss-of-function pdr1 mutants

Abstract

In the pathogenic yeast Candida glabrata, multidrug resistance is associated with the overexpression of drug efflux pumps caused by gain-of-function mutations in the CgPDR1 gene. CgPdr1p transcription factor, which activates the expression of several drug efflux transporter genes, is considered to be a promising target for compounds sensitizing the multidrug-resistant yeast cells. Here, we describe a cell-based screening system for detecting the inhibitory activity of compounds interfering with the CgPdr1p function in a heterologous genetic background of the hypersensitive Saccharomyces cerevisiae mutant strain. The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose-induced and CgPdr1p-driven overexpression of a dominant lethal pma1(D378N) allele placed under the control of the ScPDR5 promoter. The system allows rapid identification of multidrug resistance reversal agents inhibiting the CgPdr1p activity or loss-of-function Cgpdr1 mutations, and is amenable to high-throughput screening on solid or liquid media.

Fluconazole affects the alkali-metal-cation homeostasis and susceptibility to cationic toxic compounds of Candida glabrata

“…The specific uptake of potassium, as well as the export of alkali metal cations, depends on the plasma-membrane potential, which is mainly composed of the inward gradient of protons (Ramos et al , 2011; Saier et al , 2009). The proton gradient is built by the plasma H + -ATPase, which is encoded by the essential PMA1 gene in S. cerevisiae and whose orthologue CgPMA1 was identified in C. glabrata (Bairwa & Kaur, 2011; Goffa et al , 2011). The proton motive force across the plasma membrane serves as an essential source of energy for many different processes such as nutrient uptake, drug efflux and pH maintenance, and its disruption resulting in cell death is the main antifungal mechanism of certain antimycotics (Maresova et al , 2009).…”