Candida albicans is the most common fungal pathogen of AIDS patients. The infection most often involves the mucosal surfaces of the oral cavity, esophagus or vagina. In spite of the advent of triazoles as anti-fungals, management of AIDS patients with candidiasis is complicated by the development of drug-resistant C. albicans and non-albicans species. The spectrum of suitable alternatives is limited underlying the need to identify new targets, which can be exploited in the development of anti-fungals. Our approach has been to target non-mammalian phosphoproteins that are components of important signal transduction pathways of C albicans. To this end, we have identified a 2-component, histidine kinase (CaHKI) and response regulator genes (CaSSKl), both of which are essential for virulence of the organism, since knock-out strains in each gene were avirulent in comparison to parental, heterozygote and gene-reconstituted strains. The focus of this proposal will be on CaSSKl, the first response regulator isolated from C. albicans. Our reasons for emphasizing this gene are 2-fold. First, its absence results in avirulence. Second, a homologue of this gene in Schizosaccharomyces pombe provides essential adaptation functions for a number of stress conditions, including oxidative, high temperature and protein inhibitor stress. There are three specific aims in this proposal.
In specific aim 1, in vitro studies will address oxidative stress. Expression will be measured by Northern analysis and competitive PCR in parallel with post-translational studies (phosphorylation).
In specific aim 2, the virulence of CaSSKl-deleted strains (and their single-copy mates) will be evaluated in vaginal and oral models of candidiasis.
Specific aim 3 will focus upon the identification of down-stream effectors of Casskip using subtractive hybridization and the yeast 2-hybrid system. The long-term objectives of this research are to expand the number of anti-C. albicans targets and information which will lead to the development of high-throughout assays for evaluating potential inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI047047-04
Application #
6632233
Study Section
Special Emphasis Panel (ZRG1-AARR-1 (04))
Program Officer
Lambros, Chris
Project Start
2000-04-01
Project End
2005-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
4
Fiscal Year
2003
Total Cost
$312,000
Indirect Cost
Name
Georgetown University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Chauhan, Neeraj; Calderone, Richard (2008) Two-component signal transduction proteins as potential drug targets in medically important fungi. Infect Immun 76:4795-803
Walia, Aditi; Calderone, Richard (2008) The SSK2 MAPKKK of Candida albicans is required for oxidant adaptation in vitro. FEMS Yeast Res 8:287-99
Chauhan, Neeraj; Kruppa, Michael; Calderone, Richard (2007) The Ssk1p response regulator and Chk1p histidine kinase mutants of Candida albicans are hypersensitive to fluconazole and voriconazole. Antimicrob Agents Chemother 51:3747-51
Kruppa, Michael; Krom, Bastiaan P; Chauhan, Neeraj et al. (2004) The two-component signal transduction protein Chk1p regulates quorum sensing in Candida albicans. Eukaryot Cell 3:1062-5
Singh, Praveen; Chauhan, Neeraj; Ghosh, Anup et al. (2004) SKN7 of Candida albicans: mutant construction and phenotype analysis. Infect Immun 72:2390-4
Li, Dongmei; Gurkovska, Valeria; Sheridan, Michael et al. (2004) Studies on the regulation of the two-component histidine kinase gene CHK1 in Candida albicans using the heterologous lacZ reporter gene. Microbiology 150:3305-13
Kruppa, Michael; Jabra-Rizk, Mary Ann; Meiller, Timothy F et al. (2004) The histidine kinases of Candida albicans: regulation of cell wall mannan biosynthesis. FEMS Yeast Res 4:409-16
Kruppa, Michael; Goins, Tresa; Cutler, Jim E et al. (2003) The role of the Candida albicans histidine kinase [CHK1) gene in the regulation of cell wall mannan and glucan biosynthesis. FEMS Yeast Res 3:289-99
Chauhan, Neeraj; Inglis, Diane; Roman, Elvira 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 2:1018-24
Torosantucci, Antonella; Chiani, Paola; De Bernardis, Flavia et al. (2002) Deletion of the two-component histidine kinase gene (CHK1) of Candida albicans contributes to enhanced growth inhibition and killing by human neutrophils in vitro. Infect Immun 70:985-7

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