The proposed research will use Wangiella dermatitidis as a model to study cell-wall-related virulence factors among dematiaceous fungi. Our prior research has established that this fungus is an excellent model for this group of human pathogens. My emphasis on the cell-wall is dictated by the knowledge that it is a boundary between host and pathogen, an ultimate determinant of morphology, known to represent a virulence factor, and a potential target for antifungal agents. Our proposed studies are focused on two vital components of fungal cellular development and differentiation, the biosynthetic system leading to chitin localization, and the cytoskeletal system leading to actin filament formation. The tight association of actin filaments with regions of wall growth suggests they are partly responsible for site- specific insertion of chitin into the cell wall. The findings of others that actin assembly is sensitive to calcium, coupled with our new finding that polarized growth in Wangiella can be reestablished by calcium additions under the acidic conditions that induce nonpolarized growth, suggest studies with this divalent cation will be very informative. Thus, the proposed research is designed to provide important insights about the genes and enzymes of dematiaceous fungi, which are associated directly or indirectly with cell-wall construction and localization, especially the microfibrillar component chitin, and with shape regulation as related to normal growth and to dimorphic and polymorphic transitions. It is our intention to identify the components and steps in these pathways which are virulence factors and are vulnerable to antifungal intervention. The specific aims of this proposal are as follows: 1) to characterize the multiple chitin synthase genes already identified, determine their potential as antifungal target sites and establish how their expression and the function of their products affect stage specific development during vegetative polymorphism; 2) to characterize the cloned actin cytoskeletal gene of Wangiella, study its expression and determine the nature of the interactions among actin and the multiple chitin synthases and their relation to the spatial and temporal deposition of chitin leading to different phenotypes as regulated by cdc genes or by selected environmental triggers; 3) to continue to extend the concepts revealed with Wangiella to a limited number of other dematiaceous pathogens.