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Mol Microbiol. 2006 Nov;62(4):997-1013. Epub 2006 Oct 13.

Functional studies of the Ssk1p response regulator protein of Candida albicans as determined by phenotypic analysis of receiver domain point mutants.

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1
Georgetown University Medical Center, Department of Microbiology and Immunology, 3900 Reservoir Road, NW, Washington, DC 20057, USA.

Abstract

The Candida albicans response regulator protein Ssk1p regulates oxidant adaptation through the MAPK HOG1 pathway. Deletion mutants lacking SSK1 are oxidant sensitive in vitro and are killed more than wild-type (WT) cells by human neutrophils. Furthermore, the mutants are avirulent in an invasive murine model, and unable to adhere to human esophageal cells. Transcriptional profiling has indicated that approximately 25% of all changes occur in genes encoding cell wall and stress adaptation functions. In this study, we have investigated the role of amino acid residues in the Ssk1p receiver (or regulatory) domain by constructing point mutants at positions D556 (putative site of protein phosphorylation) and D513 (putative role in divalent metal binding, phosphorylation and conformational switching). For each point mutant, their sensitivity to a variety of oxidant stress conditions was assessed and correlated with in vitro phosphorylation of each Ssk1p receiver domain, phosphorylation of the Hog1p MAP kinase, and translocation to the nucleus. We show that a D556N mutant is sensitive to 5 mM H(2)O(2) or t-butyl hydroperoxide, similar to a gene knock-out ssk1 mutant, even though Hog1p is phosphorylated in the D556N mutant. To resolve this apparent paradox, we also demonstrate that Hog1p translocation to the nucleus in the D556N mutant is significantly reduced compared with WT cells (CAF2-1). In a second point mutant, D513 was changed to a lysine residue (D513K). This mutant had WT levels of resistance to peroxide, but in comparison to WT cells and the D556N mutant, morphogenesis (yeast to hyphae transition) was inhibited in 10% serum or in M-199 medium at 37 degrees C. In the D513K point mutant, constitutive phosphorylation of Hog1p was observed, suggesting that a non-conservative change (D513K) traps Ssk1p in an active conformation and therefore constitutive Hog1p phosphorylation. The inhibition of morphogenesis in D513K is related to a downregulation of the transcription factors of morphogenesis, EFG1 and CPH1. Another non-conserved point mutant (D556R) was also constructed and phenotypically was like the D513K mutant. The receiver domains of the D556N and the D513K mutants could not be appreciably phosphorylated in vitro indicating that constitutive activation of Hog1p occurs in vivo due to the inability of Ssk1p to be phosphorylated at least in the D513K mutant. We speculate that the non-conservative changes described above in Ssk1p response regulator may cause conformational changes in the Ssk1p that account for phenotype differences compared with the D556N mutant that are also Hog-independent.

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