Our research has shown that the polymorphic fungal pathogen of humans Wangiella dermatitidis has at least five chitin synthases (WdChsp), each of which is representative of a different class. Of these, newly discovered WdChs5p, a class V isozyme, requires considerable additional in-depth study, because it is the only single chitin synthase of this dematiaceous (melanized) agent of phaeohyphomycosis required for survival at 37?(3 and for full virulence in murine models of acute infection. These striking characteristics make the pathways leading to WdChs5p production and function exceptionally suitable targets for the design of antifungal drugs that are effective against dematiaceous fungi, and perhaps other pathogens with class V chitin synthases. Our rapid development of IN. dermatitidis into a molecularly tractable model for studies of chitin biosynthesis, and our numerous new and novel findings about this essential fungal process, suggest that my proposed studies of WdChsSp will provide additional important insights about chitin synthases that are wide spread in molds, but have no orthologs in Saccharomyces cerevisiae or Candida albicans. These proposed new studies will mechanistically probe how WdChsSp production is regulated by stress conditions associated with infection, why its lack of function leads to cell death at 37?C but not at 25?C, and whether its unique myosin motor-like domain contributes directly to the positional insertions of a specific cell wall chitin in stressed yeasts, hyphae or sclerotic bodies of W. dermatitidis. My proposal's revised Specific Aims are to 1) determine if the elevated transcript levels of the WdCHS5 gene detected at temperature of infection are affected by post-transcriptional modifications; 2) confirm that one or more c/s-acting elements in the 5' URS of WdCHS5 interact with trans.acting factors to up-regulate its transcription under stress conditions associated with infections; 3) establish how the product of WdCHS5 protects against lysis and death at 37?C, and document that the pathways leading to the production and function of WdChsSp and other class V chitin synthases are particularly vulnerable targets for antifungal drug design.
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