Candida albicans colonises numerous niches within humans and thus its success as a pathogen is dependent on its ability to adapt to diverse growth environments within the host. Two component signal transduction is a common mechanism by which bacteria respond to environmental stimuli and, although less common, two component-related pathways have also been characterised in fungi. Here we report the identification and characterisation of a novel two component response regulator protein in C. albicans which we have named CRR1 (Candida Response Regulator 1). Crr1 contains a receiver domain characteristic of response regulator proteins, including the conserved aspartate that receives phosphate from an upstream histidine kinase. Significantly, orthologues of CRR1 are present only in fungi belonging to the Candida CTG clade. Deletion of the C. albicans CRR1 gene, or mutation of the predicted phospho-aspartate, causes increased sensitivity of cells to the oxidising agent hydrogen peroxide. Crr1 is present in both the cytoplasm and nucleus, and this localisation is unaffected by oxidative stress or mutation of the predicted phospho-aspartate. Furthermore, unlike the Ssk1 response regulator, Crr1 is not required for the hydrogen peroxide-induced activation of the Hog1 stress-activated protein kinase pathway, or for the virulence of C. albicans in a mouse model of systemic disease. Taken together, our data suggest that Crr1, a novel response regulator restricted to the Candida CTG clade, regulates the response of C. albicans cells to hydrogen peroxide in a Hog1-independent manner that requires the function of the conserved phospho-aspartate.
References
[1]
Egger LA, Park H, Inouye M (1997) Signal Transduction via the Histidyl-Aspartyl Phosphorelay. Genes To Cells 2: 167–184.
Smith DA, Morgan BA, Quinn J (2010) Stress signalling to fungal stress-activated protein kinase pathways. FEMS Microbiology Letters 306: 1–8.
[4]
Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66: 300–372.
[5]
Reiser V, Raitt DC, Saito H (2003) Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure. J Cell Biol 161: 1035–1040.
[6]
Posas F, Wurgler-Murphy SM, Maeda T, Witten EA, Thai TC, et al. (1996) Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 “two-component” osmosensor. Cell 86: 865–875.
[7]
Posas F, Saito H (1998) Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator. Embo J 17: 1385–1394.
[8]
Buck V, Quinn J, Soto Pino T, Martin H, Saldanha J, et al. (2001) Peroxide sensors for the fission yeast stress-activated mitogen-activated protein kinase pathway. Mol Biol Cell 12: 407–419.
[9]
Quinn J, Malakasi P, Smith DA, Cheetham J, Buck V, et al. (2011) Two-component mediated peroxide sensing and signal transduction in fission yeast. Antioxid Redox Signal 15: 153–165.
[10]
Nguyen AN, Lee A, Place W, Shiozaki K (2000) Multistep phosphorelay proteins transmit oxidative stress signals to the fission yeast stress-activated protein kinase. Mol Biol Cell 11: 1169–1181.
[11]
Brown JL, Bussey H, Stewart RC (1994) Yeast Skn7p functions in a eukaryotic two-component regulatory pathway. Embo J 13: 5186–5194.
[12]
Ohmiya R, Kato C, Yamada H, Aiba H, Mizuno T (1999) A fission yeast gene (prr1+) that encodes a response regulator implicated in oxidative stress response. J Biochem (Tokyo) 125: 1061–1066.
[13]
Li S, Dean S, Li Z, Horecka J, Deschenes RJ, et al. (2002) The eukaryotic two-component histidine kinase Sln1p regulates OCH1 via the transcription factor, Skn7p. Mol Biol Cell 13: 412–424.
[14]
Morgan BA, Banks GR, Toone WM, Raitt D, Kuge S, et al. (1997) The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. Embo J 16: 1035–1044.
[15]
Chen D, Wilkinson CR, Watt S, Penkett CJ, Toone WM, et al. (2008) Multiple pathways differentially regulate global oxidative stress responses in fission yeast. Mol Biol Cell 19: 308–317.
[16]
Kruppa M, Calderone R (2006) Two-component signal transduction in human fungal pathogens. FEMS Yeast Res 6: 149–159.
[17]
Brown AJ, Haynes K, Quinn J (2009) Nitrosative and oxidative stress responses in fungal pathogenicity. Curr Opin Microbiol 12: 384–391.
[18]
Nagahashi S, Mio T, Ono N, Yamada-Okabe T, Arisawa M, et al. (1998) Isolation of CaSLN1 and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungus Candida albicans. Microbiology 144: 425–432.
[19]
Calera JA, Choi GH, Calderone RA (1998) Identification of a putative histidine kinase two-component phosphorelay gene (CaHK1) in Candida albicans. Yeast 14: 665–674.
[20]
Alex LA, Korch C, Selitrennikoff CP, Simon MI (1998) COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans. Proc Natl Acad Sci U S A 95: 7069–7073.
[21]
Srikantha T, Tsai L, Daniels K, Enger L, Highley K, et al. (1998) The two-component hybrid kinase regulator CaNIK1 of Candida albicans. Microbiology 144: 2715–2729.
[22]
Calera JA, Herman D, Calderone R (2000) Identification of YPD1, a gene of Candida albicans which encodes a two-component phosphohistidine intermediate protein. Yeast 16: 1053–1059.
[23]
Calera JA, Calderone RA (1999) Identification of a putative response regulator two-component phosphorelay gene (CaSSK1) from Candida albicans. Yeast 15: 1243–1254.
[24]
Singh P, Chauhan N, Ghosh A, Dixon F, Calderone R (2004) SKN7 of Candida albicans: mutant construction and phenotype analysis. Infect Immun 72: 2390–2394.
[25]
Chauhan N, Inglis D, Roman E, Pla J, Li D, et al. (2003) Candida albicans response regulator gene SSK1 regulates a subset of genes whose functions are associated with cell wall biosynthesis and adaptation to oxidative stress. Eukaryot Cell 2: 1018–1024.
[26]
Menon V, Li D, Chauhan N, Rajnarayanan R, Dubrovska A, et al. (2006) Functional studies of the Ssk1p response regulator protein of Candida albicans as determined by phenotypic analysis of receiver domain point mutants. Mol Microbiol 62: 997–1013.
[27]
Li D, Gurkovska V, Sheridan M, Calderone R, Chauhan N (2004) Studies on the regulation of the two-component histidine kinase gene CHK1 in Candida albicans using the heterologous lacZ reporter gene. Microbiology 150: 3305–3313.
[28]
Roman E, Nombela C, Pla J (2005) The Sho1 adaptor protein links oxidative stress to morphogenesis and cell wall biosynthesis in the fungal pathogen Candida albicans. Mol Cell Biol 25: 10611–10627.
[29]
Sherman F (1991) Getting started with yeast. Methods Enzymol 194: 3–21.
[30]
Fonzi WA, Irwin MY (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134: 717–728.
[31]
Murad AM, Lee PR, Broadbent ID, Barelle CJ, Brown AJ (2000) CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16: 325–327.
[32]
Noble SM, Johnson AD (2005) Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4: 298–309.
[33]
Dennison PMJ, Ramsdale M, Manson CL, Brown AJP (2005) Gene disruption in Candida albicans using a synthetic, codon-optimised Cre loxP system. Fungal Genet Biol 42: 737–748.
[34]
Barelle CJ, Manson CL, MacCallum DM, Odds FC, Gow NA, et al. (2004) GFP as a quantitative reporter of gene regulation in Candida albicans. Yeast 21: 333–340.
[35]
Blackwell C, Russell CL, Argimon S, Brown AJ, Brown JD (2003) Protein A-tagging for purification of native macromolecular complexes from Candida albicans. Yeast 20: 1235–1241.
[36]
Smith DA, Nicholls S, Morgan BA, Brown AJ, Quinn J (2004) A conserved stress-activated protein kinase regulates a core stress response in the human pathogen Candida albicans. Mol Biol Cell 15: 4179–4190.
[37]
Stock JB, Ninfa AJ, Stock AM (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53: 450–490.
[38]
Butler G, Rasmussen MD, Lin MF, Santos MA, Sakthikumar S, et al. (2009) Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459: 657–662.
[39]
Roman E, Cottier F, Ernst JF, Pla J (2009) Msb2 signaling mucin controls activation of Cek1 mitogen-activated protein kinase in Candida albicans. Eukaryot Cell 8: 1235–1249.
[40]
Lu JM, Deschenes RJ, Fassler JS (2003) Saccharomyces cerevisiae histidine phosphotransferase Ypd1p shuttles between the nucleus and cytoplasm for SLN1-dependent phosphorylation of Ssk1p and Skn7p. Eukaryot Cell 2: 1304–1314.
[41]
Klose KE, Weiss DS, Kustu S (1993) Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein. J Mol Biol 232: 67–78.
[42]
Calera JA, Zhao XJ, Calderone R (2000) Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans. Infect Immun 68: 518–525.
[43]
Brand A, MacCallum DM, Brown AJ, Gow NA, Odds FC (2004) Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus. Eukaryot Cell 3: 900–909.
[44]
Nikolaou E, Agrafioti I, Stumpf M, Quinn J, Stansfield I, et al. (2009) Phylogenetic diversity of stress signalling pathways in fungi. BMC Evol Biol 9: 44.
[45]
Harcus D, Nantel A, Marcil A, Rigby T, Whiteway M (2004) Transcription profiling of cyclic AMP signaling in Candida albicans. Mol Biol Cell 15: 4490–4499.
[46]
Kunze D, Melzer I, Bennett D, Sanglard D, MacCallum D, et al. (2005) Functional analysis of the phospholipase C gene CaPLC1 and two unusual phospholipase C genes, CaPLC2 and CaPLC3, of Candida albicans. Microbiology 151: 3381–3394.
[47]
Rocha CR, Schroppel K, Harcus D, Marcil A, Dignard D, et al. (2001) Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 12: 3631–3643.
[48]
Deveau A, Piispanen AE, Jackson AA, Hogan DA (2010) Farnesol induces hydrogen peroxide resistance in Candida albicans yeast by inhibiting the Ras-cyclic AMP signaling pathway. Eukaryot Cell 9: 569–577.
[49]
Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20: 133–163.
[50]
Bonhomme J, Chauvel M, Goyard S, Roux P, Rossignol T, et al. (2011) Contribution of the glycolytic flux and hypoxia adaptation to efficient biofilm formation by Candida albicans. Mol Microbiol 80: 995–1013.
[51]
Negredo A, Monteoliva L, Gil C, Pla J, Nombela C (1997) Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans. Microbiology 143: 297–302.
[52]
da Silva Dantas A, Patterson MJ, Smith DA, Maccallum DM, Erwig LP, et al. (2010) Thioredoxin regulates multiple hydrogen peroxide-induced signaling pathways in Candida albicans. Mol Cell Biol 30: 4550–4563.
