Definition of the Extracellular Proteome of Pathogenic-Phase <i>Histoplasma capsulatum</i>

Abstract

The dimorphic fungal pathogen <i>Histoplasma capsulatum</i> causes respiratory and systemic disease. Within the mammalian host, pathogenic <i>Histoplasma</i> yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to <i>Histoplasma</i> virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC−MS/MS analysis of the proteins in pathogenic-phase culture filtrates of <i>Histoplasma</i>. In addition to secreted Cbp1, the extracellular proteome of pathogenic <i>Histoplasma</i> yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp’s). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic <i>Histoplasma</i> mycelia, suggesting that these proteins are linked to <i>Histoplasma</i> pathogenesis. In addition, <i>Histoplasma</i> yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how <i>Histoplasma</i> alters the host-pathogen interaction to its advantage