The temperature-induced transition from the saprophytic mold form to the parasitic yeast form is essential for the establishment of disease by dimorphic fungi, but the genes that regulate this switch and account for yeast virulence are poorly understood. Histoplasma capsulatum and Blastomyces dermatitidis are phylogenetically the most closely related among the dimorphic fungi that cause disease worldwide and represent a growing human health problem. Development of molecular genetic tools has rendered both these fungi genetically tractable and provided the first firm evidence for the role of yeast phase-upregulated genes in virulence - CBP1 in H. capsulatum and BAD1 in B. dermatitidis. We propose here to identify and compare all of the yeast phase-upregulated genes from these two agents, then explore the functional roles of a selected subset of these genes. We hypothesize that these two fungi will exhibit both evolutionarily shared and distinct genes that regulate morphogenesis and pathogenicity. A consortium of three leading labs will combine their experience with large-scale genomics, molecular genetics, and fungal pathogenesis; the resulting collaboration will use a microarray-based approach to identify and analyze potential virulence-associated genes.
Our specific aims are to: I. Sequence the B. dermatitidis genome to approximately 3-fold coverage for comparson to H. capsulatum. II. Identify yeast phase-upregulated genes using microarrays and informatics, and prioritize genes for virulence-related studies. III. Functionally characterize in H. capsulatum and B. dermatitidis 16 to 20 of the prioritized genes by creating isogenic null mutants for investigation in animal and cell culture models of infection, exploiting lacZ and gfp reporters to monitor regulation and expression of the genes, and employing microarrays to identify other genes that are coordinately regulated. Findings from this work will provide substantial new knowledge about the biology and pathogenesis of systemic fungal diseases and may identify among conserved genes many potential new drug targets.
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