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Eukaryotic Cell, October 2003, p. 1018-1024, Vol. 2, No. 5
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.5.1018-1024.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Candida albicans Response Regulator Gene SSK1 Regulates a Subset of Genes Whose Functions Are Associated with Cell Wall Biosynthesis and Adaptation to Oxidative Stress

Neeraj Chauhan,1 Diane Inglis,2 Elvira Roman,3 Jesus Pla,3 Dongmei Li,1 Jose A. Calera,4 and Richard Calderone1*

Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, District of Columbia 20057,1 Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California,2 Departamento de Microbiología II, Universidad Complutense de Madrid, Madrid,3 Departamento de Microbiología, Universidad de Salamanca, Salamanca, Spain4

Received 23 July 2003/ Accepted 29 July 2003

Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30°C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Georgetown University School of Medicine, 312 SE Med-Dent Building, 3900 Reservoir Rd., NW, Washington, DC 20057. Phone: (202) 687-1137. Fax: (202) 687-1800. E-mail: calderor{at}georgetown.edu.

Eukaryotic Cell, October 2003, p. 1018-1024, Vol. 2, No. 5
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.5.1018-1024.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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