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Current Genetics

, Volume 59, Issue 3, pp 91–106 | Cite as

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

  • Guadalupe Gutiérrez-Escobedo
  • Emmanuel Orta-Zavalza
  • Irene Castaño
  • Alejandro De Las PeñasEmail author
Research Article

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 gsh1pro2-4 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 gsh1pro2-4 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.

Keywords

Candida glabrata Glutathione Cd tolerance PRO2 suppressor Oxidative stress Catalase Thioredoxin 

Notes

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.

References

  1. 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 15:385–396PubMedCrossRefGoogle Scholar
  2. Anderson ME (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113:548–555PubMedCrossRefGoogle Scholar
  3. Ausubel FM (1992) Short protocols in molecular biology: a compendium of methods from Current protocols in molecular biology. Greene, New YorkGoogle Scholar
  4. Avery AM, Avery SV (2001) Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases. J Biol Chem 276:33730–33735PubMedCrossRefGoogle Scholar
  5. 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 275:13259–13265PubMedCrossRefGoogle Scholar
  6. Byrne KP, Wolfe KH (2005) The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res 15:1456–1461PubMedCrossRefGoogle Scholar
  7. Calvin NM, Hanawalt PC (1988) High-efficiency transformation of bacterial cells by electroporation. J Bacteriol 170:2796–2801Google Scholar
  8. 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 54:439–461PubMedCrossRefGoogle Scholar
  9. Castano I, Kaur R, Pan S et al (2003) Tn7-based genome-wide random insertional mutagenesis of Candida glabrata. Genome Res 13:905–915PubMedCrossRefGoogle Scholar
  10. 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 55:1246–1258PubMedCrossRefGoogle Scholar
  11. 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 145:75–83PubMedGoogle Scholar
  12. Cormack BP, Falkow S (1999) Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata. Genetics 151:979–987Google Scholar
  13. Cormack BP, Ghori N, Falkow S (1999) An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science 285:578–582PubMedCrossRefGoogle Scholar
  14. 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 7:814–825PubMedCrossRefGoogle Scholar
  15. 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 17:2245–2258CrossRefGoogle Scholar
  16. 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 40:1298–1302PubMedCrossRefGoogle Scholar
  17. 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 308:866–870Google Scholar
  18. Fernandes AP, Holmgren A (2004) Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system. Antioxid Redox Signal 6:63–74PubMedCrossRefGoogle Scholar
  19. Fidel PL, Cutright JL, Tait L, Sobel JD (1996) A murine model of Candida glabrata vaginitis. J Infect Dis 173:425–431Google Scholar
  20. Gasch AP (2007) Comparative genomics of the environmental stress response in ascomycete fungi. Yeast 24:961–976PubMedCrossRefGoogle Scholar
  21. 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 74:527–534Google Scholar
  22. 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 29:511–515PubMedCrossRefGoogle Scholar
  23. 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 8:1699–1707PubMedGoogle Scholar
  24. 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 253:893–898PubMedCrossRefGoogle Scholar
  25. Halliwell B, Gutteridge JMC (2007) Free radicals in biology and medicine. Oxford University Press, OxfordGoogle Scholar
  26. Hauser M, Donhardt AM, Barnes D, Naider F, Becker JM (2000) Enkephalins are transported by a novel eukaryotic peptide uptake system. J Biol Chem 275:3037–3041PubMedCrossRefGoogle Scholar
  27. 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 5:157–167PubMedCrossRefGoogle Scholar
  28. Higgins DG, Thompson JD, Gibson TJ (1996) Using CLUSTAL for multiple sequence alignments. Methods Enzymol 266:383–402PubMedCrossRefGoogle Scholar
  29. 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 148:3705–3713PubMedGoogle Scholar
  30. 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):61–67PubMedGoogle Scholar
  31. 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:B34PubMedGoogle Scholar
  32. 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 104:7628–7633PubMedCrossRefGoogle Scholar
  33. 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 219:187–194PubMedCrossRefGoogle Scholar
  34. 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 30:2044–2056PubMedCrossRefGoogle Scholar
  35. 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:E2PubMedCrossRefGoogle Scholar
  36. 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 274:16040–16046PubMedCrossRefGoogle Scholar
  37. 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 94:42–47PubMedCrossRefGoogle Scholar
  38. Li L, Redding S, Dongari-Bagtzoglou A (2007) Candida glabrata: an emerging oral opportunistic pathogen. J Dent Res 86:204–215PubMedCrossRefGoogle Scholar
  39. Lopez-Mirabal HR, Winther JR (2008) Redox characteristics of the eukaryotic cytosol. Biochim Biophys Acta 1783:629–640PubMedCrossRefGoogle Scholar
  40. 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 9:1081–1091PubMedCrossRefGoogle Scholar
  41. Mansour MK, Levitz SM (2002) Interactions of fungi with phagocytes. Curr Opin Microbiol 5:359–365PubMedCrossRefGoogle Scholar
  42. Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760PubMedCrossRefGoogle Scholar
  43. Muller EG (1991) Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle. J Biol Chem 266:9194–9202PubMedGoogle Scholar
  44. 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 63:113–121PubMedCrossRefGoogle Scholar
  45. 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 138:797–804PubMedCrossRefGoogle Scholar
  46. 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 11:3491–3499PubMedGoogle Scholar
  47. Paris S, Wysong D, Debeaupuis JP, Shibuya K, Philippe B, Diamond RD, Latge JP (2003) Catalases of Aspergillus fumigatus. Infect Immun 71:3551–3562PubMedCrossRefGoogle Scholar
  48. Penninckx MJ (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res 2:295–305PubMedGoogle Scholar
  49. Penninckx MJ, Elskens MT (1993) Metabolism and functions of glutathione in micro-organisms. Adv Microb Physiol 34:239–301PubMedCrossRefGoogle Scholar
  50. Pfaller MA, Diekema DJ (2010) Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36:1–53PubMedCrossRefGoogle Scholar
  51. 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 13:1072–1076PubMedCrossRefGoogle Scholar
  52. Rauser WE (1990) Phytochelatins. Annu Rev Biochem 59:61–86PubMedCrossRefGoogle Scholar
  53. Rauser WE (1995) Phytochelatins and related peptides. Structure, biosynthesis, and function. Plant Physiol 109:1141–1149PubMedCrossRefGoogle Scholar
  54. Roetzer A, Gregori C, Jennings AM et al (2008) Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors. Mol Microbiol 69:603–620PubMedCrossRefGoogle Scholar
  55. Roetzer A, Gratz N, Kovarik P, Schuller C (2010) Autophagy supports Candida glabrata survival during phagocytosis. Cell Microbiol 12:199–216PubMedCrossRefGoogle Scholar
  56. Roetzer A, Klopf E, Gratz N et al (2011) Regulation of Candida glabrata oxidative stress resistance is adapted to host environment. FEBS Lett 585:319–327PubMedCrossRefGoogle Scholar
  57. Saijo T, Miyazaki T, Izumikawa K et al (2010) Skn7p is involved in oxidative stress response and virulence of Candida glabrata. Mycopathologia 169:81–90PubMedCrossRefGoogle Scholar
  58. Seider K, Brunke S, Schild L et al (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation. J Immunol 187:3072–3086PubMedCrossRefGoogle Scholar
  59. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, USAGoogle Scholar
  60. Sipos K, Lange H, Fekete Z, Ullmann P, Lill R, Kispal G (2002) Maturation of cytosolic iron-sulfur proteins requires glutathione. J Biol Chem 277:26944–26949PubMedCrossRefGoogle Scholar
  61. 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 276:7011–7016PubMedCrossRefGoogle Scholar
  62. Stephen DW, Jamieson DJ (1996) Glutathione is an important antioxidant molecule in the yeast Saccharomyces cerevisiae. FEMS Microbiol Lett 141:207–212PubMedCrossRefGoogle Scholar
  63. Temple MD, Perrone GG, Dawes IW (2005) Complex cellular responses to reactive oxygen species. Trends Cell Biol 15:319–326PubMedCrossRefGoogle Scholar
  64. 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 71:2643–2655PubMedCrossRefGoogle Scholar
  65. 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 101:6564–6569PubMedCrossRefGoogle Scholar
  66. 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 581:3598–3607PubMedCrossRefGoogle Scholar
  67. Tomenchok DM, Brandriss MC (1987) Gene-enzyme relationships in the proline biosynthetic pathway of Saccharomyces cerevisiae. J Bacteriol 169:5364–5372PubMedGoogle Scholar
  68. Veeravalli K, Boyd D, Iverson BL, Beckwith J, Georgiou G (2011) Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways. Nat Chem Biol 7:101–105PubMedCrossRefGoogle Scholar
  69. 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 66:1953–1961PubMedGoogle Scholar
  70. 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 157:484–495PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Guadalupe Gutiérrez-Escobedo
    • 1
  • Emmanuel Orta-Zavalza
    • 1
  • Irene Castaño
    • 1
  • Alejandro De Las Peñas
    • 1
    Email author
  1. 1.IPICYT, Camino a la Presa San José 2055, División de Biología MolecularInstituto Potosino de Investigación Científica y TecnológicaSan Luis PotosíMéxico

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