| Histoplasma capsulatum is a dimorphic, pathogenic fungus that is the causative agent of histoplasmosis. Despite having no known requirement for infecting a mammalian host as part of its lifecycle, Histoplasma capsulatum is well adapted to cause mammalian disease. The mold-to-yeast transition is essential for virulence and is associated with major changes in cellular metabolism. In response to phase transition, H. capsulatum changes the mRNA level of genes associated with nutrient acquisition, thermotolerance, and stress response, as well as the aptly named yeast-phase-specific genes. The YPS3 locus encodes a protein that, beyond being yeast phase specific, is also produced exclusively in the most virulent H. capsulatum strains. Here we describe our observations on the conservation, localization and virulence implications of the Histoplasma capsulatum YPS3 gene.; We analyzed 32 strains and variants of H. capsulatum and discovered YPS3 loci in all strains surveyed, although protein was only produced in a fraction of these. This analysis revealed an intragenic region of variable length comprised of 5--6 amino acid tandem repeats. Surveyed strains had between 2--20 copies of the tandem repeat. The Yps3 protein is both secreted and cell wall localized. We used flow cytometry and immunofluorescence microscopy to reveal that the cell wall localized Yps3p is surface exposed. Polysaccharide binding assays and enzymatic digestions revealed the mature Yps3p protein is secreted from the cell and binds back to surface exposed cell wall polysaccharide chitin. We designed RNAi vectors to interfere with RNA production from the YPS3 locus, and greatly reduced Yps3p protein levels. We analyzed YPS3 RNAi mutants for in vitro growth, infection in the murine macrophage like cell line RAW 264.7, and in a mouse model of infection. Despite having no effect on in vitro growth or in a macrophage like cell line, the RNAi mutants were attenuated in organ burden in lungs, livers, and spleens during mouse infection. This attenuation was exacerbated in the peripheral tissues. Taken together, this research expands our understanding of subcellular protein localization and cell wall architecture, and suggests a role for Yps3p in mammalian virulence. |
