Candida albicans causes more serious infections in humans that any other fungus. The C. albicans genome program is making steady progress and it is expected that > 6000 genes will be sequenced within the next year. The function of many of these genes can be studied by gene disruption and phenotypic analysis, but essential genes cannot be disrupted without loss of viability. The overall goals of this project are to: 1) develop new approaches for studying essential C. albicans genes using two secretion pathway genes as models and 2) use these approaches to study intracellular transport and secretion of two virulence associated C. albicans proteins. In Saccharomyces cerevisiae, SEC4 and YPT1 encode small ras-like GTPases that are required, respectively for fusion of post-Golgi secretory vesicles to the plasma membrane and for ER-to-Golgi protein transport. The SEC4 and YPT1 genes of C. albicans have been cloned and sequenced. When gene disruption experiment suggested that C. albicans SEC4 was essential, it was found that over expressing a mutant sec4 allele similar to those encoding dominant inhibitors of other ras-like GTPases inhibited growth, protein secretion and fusion of secretory vesicles to the plasma membrane in C. albicans. These results demonstrated the feasibility of using molecular approaches to study essential C. albicans genes.
Aim 1 is to i) generate C. albicans strains with temperature sensitive and/or inducible dominant-negative sec4 mutations and ii) determine if double-stranded RNAs can block expression of SEC4 and other C. albicans genes.
Aim 2 will define the functions of C. albicans SEC4. The C. albicans strains from Aim 1 will be tested for growth an survival, morphology and germ tube formation, ultrastructure, and the ability to transport and secrete aspartyl protease and phospholipase B.
Aim 3 will generate C. albicans strains with loss-of-function ypt1 mutations and to use these mutants to define the functions of C. albicans YPT1.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI047442-04
Application #
6632285
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Duncan, Rory A
Project Start
2000-04-01
Project End
2005-03-31
Budget Start
2003-04-01
Budget End
2005-03-31
Support Year
4
Fiscal Year
2003
Total Cost
$253,750
Indirect Cost
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Kayingo, Gerald; Martins, António; Andrie, Rachael et al. (2009) A permease encoded by STL1 is required for active glycerol uptake by Candida albicans. Microbiology 155:1547-57
Mao, Yuxin; Zhang, Zimei; Gast, Charles et al. (2008) C-terminal signals regulate targeting of glycosylphosphatidylinositol-anchored proteins to the cell wall or plasma membrane in Candida albicans. Eukaryot Cell 7:1906-15
Lee, Samuel A; Jones, Jason; Khalique, Zachary et al. (2007) A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4. FEMS Yeast Res 7:973-85
Lee, Samuel A; Khalique, Zachary; Gale, Cheryl A et al. (2005) Intracellular trafficking of fluorescently tagged proteins associated with pathogenesis in Candida albicans. Med Mycol 43:423-30
Kayingo, Gerald; Wong, Brian (2005) The MAP kinase Hog1p differentially regulates stress-induced production and accumulation of glycerol and D-arabitol in Candida albicans. Microbiology 151:2987-99
Lee, Samuel A; Wormsley, Steven; Kamoun, Sophien et al. (2003) An analysis of the Candida albicans genome database for soluble secreted proteins using computer-based prediction algorithms. Yeast 20:595-610
Mao, Yuxin; Zhang, Zimei; Wong, Brian (2003) Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans. Mol Microbiol 50:1617-28
Yeo, Siew Fah; Wong, Brian (2002) Current status of nonculture methods for diagnosis of invasive fungal infections. Clin Microbiol Rev 15:465-84
Lee, S A; Mao, Y; Zhang, Z et al. (2001) Overexpression of a dominant-negative allele of YPT1 inhibits growth and aspartyl protease secretion in Candida albicans. Microbiology 147:1961-70