Histoplasma capsulatum is a pathogenic fungus of worldwide distribution and the cause of a disease endemic to the midwestern United States. The pathogenesis of histoplasmosis is poorly understood and to date no specific virulence factors have been identified. Although, not an obligate intracellular pathogen, H. capsulatum is well suited for survival in alveolar macrophages. The organism circumvents many of the formally microbicidal mechanism of macrophages and is able to proliferate in the hostile environment of the phagolysosome. Our laboratory has reported that H. capsulatum resides in phagolysosomes having a pH 1 to 2 units higher than vesicles containing other particles. In vitro H. capsulatum also alkalinizes its environment, and we have determined that this is due to the release of spermine, a low molecular weight polyamine. We hypothesize that spermine acts as a buffering agent to keep the pH near neutrality in the normally acidic phagolysosome. This proposal applies a new molecular technique to isolate random, genetically marked mutations in H. capsulatum. Mutants in spermine production will be identified by several methods and tested for the ability to raise the pH in H. capsulatum -containing vesicles. Spermine biosynthesis mutants will be examined for virulence in vitro and in an animal model of histoplasmosis. The mutated sequences will also be used to clone corresponding wildtype genes from a H. capsulatum genomic library. Characterization of these clones and phenotypic complementation of the mutant will confirm any correlations between spermine biosynthesis genes and Histoplasma virulence.
