Histoplasma capsulatum is one of the classic [unreadable]dimorphic[unreadable] fungal pathogens, undergoing a temperature-induced transition from a mold form that grows in soil to a yeast form that establishes infection in lung macrophages. All of the dimorphic fungi cause the most severe disease in immunocompromised patients, but they are primary pathogens that can cause serious problems in hosts lacking any demonstrable immune defect. This is a competing continuation of a grant in which the last funding period was focused on identifying and characterizing Histoplasma yeast phase-specific genes, proteins, and polysaccharides. Two of these, CBP (a secreted protein) and &#945;-(1,3)-glucan (a cell wall polysaccharide), became the first genetically proven virulence factors of H. capsulatum. The first two portions of the current research plan are designed to expand the understanding of how these virulence factors work and how they are regulated: Specific Aim 1. Define the function and regulation of CBP. Solving the three-dimensional structure of CBP revealed a surprising similarity to saposin B, a mammalian protein involved in membrane processing and antigen presentation. The studies in this Aim are designed to characterize the functional relationship between CBP and saposins, as well as to identify regulators of CBP1 gene expression. Specific Aim 2. Identify the genes involved in &#945;-(1,3)-glucan production. Previous work has demonstrated that an outer layer of alpha-(1,3)-glucan helps mask the organisms from immune recognition. Because this polysaccharide is not synthesized by mammalian cells, the genes involved in its production encode potential drug targets. The experiments in this Aim are focused on identifying and characterizing the genes and proteins involved in the synthesis, regulation, and processing of alpha-(1,3)-glucan. The third portion of the research plan focuses on three newly discovered virulence-associated genes that, like alpha-(1,3)-glucan and CBP, are (i) upregulated in the yeast phase and (ii) have roles in either the initial interaction of Histoplasma with host cells or in the ability of the organisms to survive and proliferate intracellularly: Specific Aim 3. Characterize the role of other putative virulence factors in histoplasmosis. Two of these virulence-associated genes contribute to the ability of H. capsulatum to adhere to macrophages. The third gene encodes a secreted phospholipase that is important for proliferation in macrophages and may work synergistically with CBP.