Abstract
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 (γ-glutamyl-cysteine synthetase) and Gsh2 (glutathione synthetase) enzymes. We first screened for suppressor mutations that would allow growth in the absence of GSH1 (gsh1∆ background) and found a single point mutation in PRO2 (pro2-4), a gene that encodes a γ-glutamyl phosphate reductase and catalyzes the second step in the biosynthesis of proline. We demonstrate that GSH is important in the OSR since the gsh1∆ pro2-4 and gsh2∆ 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 gsh1∆ pro2-4 and gsh2∆ 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.
Similar content being viewed by others
References
Al-Lahham A, Rohde V, Heim P et al (1999) Biosynthesis of phytochelatins in the fission yeast. Phytochelatin synthesis: a second role for the glutathione synthetase gene of Schizosaccharomyces pombe. Yeast 15:385–396
Anderson ME (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113:548–555
Ausubel FM (1992) Short protocols in molecular biology: a compendium of methods from Current protocols in molecular biology. Greene, New York
Avery AM, Avery SV (2001) Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases. J Biol Chem 276:33730–33735
Bourbouloux A, Shahi P, Chakladar A, Delrot S, Bachhawat AK (2000) Hgt1p, a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae. J Biol Chem 275:13259–13265
Byrne KP, Wolfe KH (2005) The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res 15:1456–1461
Calvin NM, Hanawalt PC (1988) High-efficiency transformation of bacterial cells by electroporation. J Bacteriol 170:2796–2801
Carmel-Harel O, Storz G (2000) Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and saccharomyces cerevisiae responses to oxidative stress. Annu Rev Microbiol 54:439–461
Castano I, Kaur R, Pan S et al (2003) Tn7-based genome-wide random insertional mutagenesis of Candida glabrata. Genome Res 13:905–915
Castano I, Pan SJ, Zupancic M, Hennequin C, Dujon B, Cormack BP (2005) Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata. Mol Microbiol 55:1246–1258
Chaudhuri B, Ingavale S, Bachhawat AK (1997) apd1 + , a gene required for red pigment formation in ade6 mutants of Schizosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role for glutathione and a glutathione-conjugate pump. Genetics 145:75–83
Cormack BP, Falkow S (1999) Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata. Genetics 151:979–987
Cormack BP, Ghori N, Falkow S (1999) An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science 285:578–582
Cuellar-Cruz M, Briones-Martin-del-Campo M, Canas-Villamar I, Montalvo-Arredondo J, Riego-Ruiz L, Castano I, De Las Penas A (2008) High resistance to oxidative stress in the fungal pathogen Candida glabrata is mediated by a single catalase, Cta1p, and is controlled by the transcription factors Yap1p, Skn7p, Msn2p, and Msn4p. Eukaryot Cell 7:814–825
De Las PenasA, Pan SJ, Castano I, Alder J, Cregg R, Cormack BP (2003) Virulence-related surface glycoproteins in the yeast pathogen Candida glabrata are encoded in subtelomeric clusters and subject to RAP1- and SIR-dependent transcriptional silencing. Genes Dev 17:2245–2258
Diekema DJ, Messer SA, Brueggemann AB, Coffman SL, Doern GV, Herwaldt LA, Pfaller MA (2002) Epidemiology of candidemia: 3-year results from the emerging infections and the epidemiology of Iowa organisms study. J Clin Microbiol 40:1298–1302
Domergue R, Castano I, De Las Penas A, Zupancic M, Lockatell V, Hebel JR, Johnson D, Cormack BP (2005) Nicotinic acid limitation regulates silencing of Candida adhesins during UTI. Science 308:866–870
Fernandes AP, Holmgren A (2004) Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system. Antioxid Redox Signal 6:63–74
Fidel PL, Cutright JL, Tait L, Sobel JD (1996) A murine model of Candida glabrata vaginitis. J Infect Dis 173:425–431
Gasch AP (2007) Comparative genomics of the environmental stress response in ascomycete fungi. Yeast 24:961–976
Gietz RD, Sugino A (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534
Grant CM, MacIver FH, Dawes IW (1996) Glutathione is an essential metabolite required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. Curr Genet 29:511–515
Grant CM, MacIver FH, Dawes IW (1997) Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine. Mol Biol Cell 8:1699–1707
Grant CM, Perrone G, Dawes IW (1998) Glutathione and catalase provide overlapping defenses for protection against hydrogen peroxide in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 253:893–898
Halliwell B, Gutteridge JMC (2007) Free radicals in biology and medicine. Oxford University Press, Oxford
Hauser M, Donhardt AM, Barnes D, Naider F, Becker JM (2000) Enkephalins are transported by a novel eukaryotic peptide uptake system. J Biol Chem 275:3037–3041
Heeren G, Jarolim S, Laun P et al (2004) The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway. FEMS Yeast Res 5:157–167
Higgins DG, Thompson JD, Gibson TJ (1996) Using CLUSTAL for multiple sequence alignments. Methods Enzymol 266:383–402
Hwang CS, Rhie GE, Oh JH, Huh WK, Yim HS, Kang SO (2002) Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is required for the protection of Candida albicans against oxidative stresses and the expression of its full virulence. Microbiology 148:3705–3713
Izawa S, Inoue Y, Kimura A (1996) Importance of catalase in the adaptive response to hydrogen peroxide: analysis of acatalasaemic Saccharomyces cerevisiae. Biochem J 320(Pt 1):61–67
Jaspers C, Penninckx M, Wiame JM (1980) Glutathione metabolism and the transport of amino acids in Saccharomyces cerevisiae. The gamma-glutamyltranspeptidase [proceedings]. Arch Int Physiol Biochim 88:B34
Kaur R, Ma B, Cormack BP (2007) A family of glycosylphosphatidylinositol-linked aspartyl proteases is required for virulence of Candida glabrata. Proc Natl Acad Sci USA 104:7628–7633
Kumar C, Sharma R, Bachhawat AK (2003) Utilization of glutathione as an exogenous sulfur source is independent of gamma-glutamyl transpeptidase in the yeast Saccharomyces cerevisiae: evidence for an alternative glutathione degradation pathway. FEMS Microbiol Lett 219:187–194
Kumar C, Igbaria A, D’Autreaux B et al (2011) Glutathione revisited: a vital function in iron metabolism and ancillary role in thiol-redox control. EMBO J 30:2044–2056
Kuwayama H, Obara S, Morio T, Katoh M, Urushihara H, Tanaka Y (2002) PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. Nucleic Acids Res 30:E2
Lee J, Godon C, Lagniel G, Spector D, Garin J, Labarre J, Toledano MB (1999) Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J Biol Chem 274:16040–16046
Li ZS, Lu YP, Zhen RG, Szczypka M, Thiele DJ, Rea PA (1997) A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium. Proc Natl Acad Sci USA 94:42–47
Li L, Redding S, Dongari-Bagtzoglou A (2007) Candida glabrata: an emerging oral opportunistic pathogen. J Dent Res 86:204–215
Lopez-Mirabal HR, Winther JR (2008) Redox characteristics of the eukaryotic cytosol. Biochim Biophys Acta 1783:629–640
Luikenhuis S, Perrone G, Dawes IW, Grant CM (1998) The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species. Mol Biol Cell 9:1081–1091
Mansour MK, Levitz SM (2002) Interactions of fungi with phagocytes. Curr Opin Microbiol 5:359–365
Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760
Muller EG (1991) Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle. J Biol Chem 266:9194–9202
Murakami CJ, Burtner CR, Kennedy BK, Kaeberlein M (2008) A method for high-throughput quantitative analysis of yeast chronological life span. J Gerontol A Biol Sci Med Sci 63:113–121
Mutoh N, Kawabata M, Kitajima S (2005) Effects of four oxidants, menadione, 1-chloro-2,4-dinitrobenzene, hydrogen peroxide and cumene hydroperoxide, on fission yeast Schizosaccharomyces pombe. J Biochem 138:797–804
Ortiz DF, Kreppel L, Speiser DM, Scheel G, McDonald G, Ow DW (1992) Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter. EMBO J 11:3491–3499
Paris S, Wysong D, Debeaupuis JP, Shibuya K, Philippe B, Diamond RD, Latge JP (2003) Catalases of Aspergillus fumigatus. Infect Immun 71:3551–3562
Penninckx MJ (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res 2:295–305
Penninckx MJ, Elskens MT (1993) Metabolism and functions of glutathione in micro-organisms. Adv Microb Physiol 34:239–301
Pfaller MA, Diekema DJ (2010) Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36:1–53
Presterl E, Daxbock F, Graninger W, Willinger B (2007) Changing pattern of candidemia 2001–2006 and use of antifungal therapy at the University Hospital of Vienna, Austria. Clin Microbiol Infect 13:1072–1076
Rauser WE (1990) Phytochelatins. Annu Rev Biochem 59:61–86
Rauser WE (1995) Phytochelatins and related peptides. Structure, biosynthesis, and function. Plant Physiol 109:1141–1149
Roetzer A, Gregori C, Jennings AM et al (2008) Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors. Mol Microbiol 69:603–620
Roetzer A, Gratz N, Kovarik P, Schuller C (2010) Autophagy supports Candida glabrata survival during phagocytosis. Cell Microbiol 12:199–216
Roetzer A, Klopf E, Gratz N et al (2011) Regulation of Candida glabrata oxidative stress resistance is adapted to host environment. FEBS Lett 585:319–327
Saijo T, Miyazaki T, Izumikawa K et al (2010) Skn7p is involved in oxidative stress response and virulence of Candida glabrata. Mycopathologia 169:81–90
Seider K, Brunke S, Schild L et al (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation. J Immunol 187:3072–3086
Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, USA
Sipos K, Lange H, Fekete Z, Ullmann P, Lill R, Kispal G (2002) Maturation of cytosolic iron-sulfur proteins requires glutathione. J Biol Chem 277:26944–26949
Spector D, Labarre J, Toledano MB (2001) A genetic investigation of the essential role of glutathione: mutations in the proline biosynthesis pathway are the only suppressors of glutathione auxotrophy in yeast. J Biol Chem 276:7011–7016
Stephen DW, Jamieson DJ (1996) Glutathione is an important antioxidant molecule in the yeast Saccharomyces cerevisiae. FEMS Microbiol Lett 141:207–212
Temple MD, Perrone GG, Dawes IW (2005) Complex cellular responses to reactive oxygen species. Trends Cell Biol 15:319–326
Thompson LJ, Merrell DS, Neilan BA, Mitchell H, Lee A, Falkow S (2003) Gene expression profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene expression. Infect Immun 71:2643–2655
Thorpe GW, Fong CS, Alic N, Higgins VJ, Dawes IW (2004) Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stress-response genes. Proc Natl Acad Sci USA 101:6564–6569
Toledano MB, Kumar C, Le Moan N, Spector D, Tacnet F (2007) The system biology of thiol redox system in Escherichia coli and yeast: differential functions in oxidative stress, iron metabolism and DNA synthesis. FEBS Lett 581:3598–3607
Tomenchok DM, Brandriss MC (1987) Gene-enzyme relationships in the proline biosynthetic pathway of Saccharomyces cerevisiae. J Bacteriol 169:5364–5372
Veeravalli K, Boyd D, Iverson BL, Beckwith J, Georgiou G (2011) Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways. Nat Chem Biol 7:101–105
Wysong DR, Christin L, Sugar AM, Robbins PW, Diamond RD (1998) Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene. Infect Immun 66:1953–1961
Yadav AK, Desai PR, Rai MN, Kaur R, Ganesan K, Bachhawat AK (2011) Glutathione biosynthesis in the yeast pathogens Candida glabrata and Candida albicans: essential in C. glabrata, and essential for virulence in C. albicans. Microbiology 157:484–495
Acknowledgments
We thank Lina Riego, Marcela Briones and Jacqueline Juárez for helpful discussions. This work was funded by a Consejo Nacional de Ciencia y Tecnología (CONACYT) fellowship to G.G.E. (48580) and E.O.Z. (233455). This work was funded by a CONACYT grant no. CB-2010-01-153929 to A.D.L.P and grant no. CB-2010-01-151517 to I.C.N.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by B. Cormack.
Rights and permissions
About this article
Cite this article
Gutiérrez-Escobedo, G., Orta-Zavalza, E., Castaño, I. et al. Role of glutathione in the oxidative stress response in the fungal pathogen Candida glabrata . Curr Genet 59, 91–106 (2013). https://doi.org/10.1007/s00294-013-0390-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-013-0390-1