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.
