Role of glutathione in the oxidative stress response in the fungal pathogen Candida glabrata

Candida glabrata, an opportunistic fungal pathogen, accounts for 18-26 % of all Candida systemic infections in the US. C. glabrata has a robust oxidative stress response (OSR) and in this work we characterized the role of glutathione (GSH), an essential tripeptide-like thiol-containing molecule required to keep the redox homeostasis and in the detoxification of metal ions. GSH is synthesized from glutamate, cysteine, and glycine by the sequential action of Gsh1 (gamma-glutamyl-cysteine synthetase) and Gsh2 (glutathione synthetase) enzymes. We first screened for suppressor mutations that would allow growth in the absence of GSH1 (gsh1a dagger background) and found a single point mutation in PRO2 (pro2-4), a gene that encodes a gamma-glutamyl phosphate reductase and catalyzes the second step in the biosynthesis of proline. We demonstrate that GSH is important in the OSR since the gsh1a dagger pro2-4 and gsh2a dagger mutant strains are more sensitive to oxidative stress generated by H2O2 and menadione. GSH is also required for Cadmium tolerance. In the absence of Gsh1 and Gsh2, cells show decreased viability in stationary phase. Furthermore, C. glabrata does not contain Saccharomyces cerevisiae high affinity GSH transporter ortholog, ScOpt1/Hgt1, however, our genetic and biochemical experiments show that the gsh1a dagger pro2-4 and gsh2a dagger mutant strains are able to incorporate GSH from the medium. Finally, GSH and thioredoxin, which is a second redox system in the cell, are not essential for the catalase-independent adaptation response to H2O2.