Histoplasma capsulatum is a complex pathogen that can cause a wide variety of syndromes, depending on the pathway of infection. Understanding the spectrum of histoplasmosis demands a thorough approach to answering a central biological question: How does H. capsulatum survive and proliferate within host cells? Biochemical, cell biological, and molecular genetic studies will be combined over the next 5 years with the following 3 specific aims: I. Understanding the molecular basis of the rough/smooth yeast phenotypic variation. This spontaneous variation correlates with the loss of cell wall 1-(1.3)-glucan, which will be studied in terms of its regulation and its relationship to virulence. The primary genetic strategy is to identify genes involved in this variation by complementation cloning in H. capsulatum, using a shuttle plasmid to transform a genomic library from the wild-type (rough) strain into an isogenic variant (smooth) strain. II. Defining the role and regulation of a calcium-binding protein (CBP) in calcium acquisition and virulence. CBP is a major secreted product of the yeast form H. capsulatum and correlates with the yeast's ability to grow in calcium-limited conditions. The native CBP I gene will be disrupted using a strategy that employs two genetic markers to enrich for allelic replacement events. In addition, a laZ-based reporter system will be exploited to identify trans-acting regulators that modulate CPB 1 expression. III. Generating physically marked mutants in virulence- associated phenotypes. This takes advantage of the technique of restriction enzyme-mediated integration (REMI) to generate a bank of insertion mutants of H. capsulatum. These will be screened/selected for defects in phenotypes that are already suspected to be associated with virulence.
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