Identification and Characterization of Histoplasma capsulatum extracellular proteins and their roles in virulence

Histoplasma capsulatum is the most common cause of endemic mycosis in the world and is endemic to the Midwestern U.S. Histoplasma commonly resides within soil as conidia-producing mycelia, but upon temperature shift to 37°C it undergoes a morphological shift to yeast that parasitize phagocytic cells and cause disease. The mechanisms allowing Histoplasma to circumvent the innate immune response and cause disease are largely unknown, however all known virulence determinants are extracellular factors that are predicted to interact with the host immune system and promote Histoplasma survival. The purpose of this dissertation research is to identify the extracellular proteins released by pathogenic-phase Histoplasma and to characterize the involvement of these proteins in Histoplasma pathogenesis. Utilizing shotgun proteomics we identified the 33 most abundant extracellular proteins, including factors from several diverse functional groups. The groups included proteins involved in cell wall biosynthesis, oxidative stress defense, chaperone activity and proteins of unknown function (Cfp4). We identified proteins with potential roles in virulence by determining which were expressed primarily by Histoplasma cells in the pathogenic-phase and that those that are expressed during residence in macrophages and during lung infections. From this, we prioritized a extracellular superoxide dismutase (Sod3), extracellular catalase (CatB) and a protein of unknown function (Cfp4) for further analysis and Histoplasma strains depleted of these proteins were created to functionally characterize these factors. We demonstrated that Sod3 is required for Histoplasma survival against neutrophils and activated macrophages by protecting Histoplasma against host-derived superoxide. The Sod3 protein is both secreted and attached to the yeast cell wall by a putative GPI- anchor. At the cell surface, Sod3 specifically protects Histoplasma against extracellular superoxide such as that produced by host phagocytes. Sod3 is required for the survival of Histoplasma in vivo and is required for disease causation. In contrast, the CatB protein is not required for Histoplasma survival in macrophages or in vivo, despite being required to protect yeast against hydrogen peroxide in vitro. To determine if Sod3 and CatB act synergistically protect Histoplasma against host-derived reactive oxygen we generated the first double deletion mutant in Histoplasma. Our results show that Sod3 but not CatB provide protection to Histoplasma. To determine if the non-essentiality of CatB for virulence results from redundancy with additional catalases, we investigated the contributions of an intracellular catalase, CatP. We also determined that Cfp4, is not required for Histoplasma virulence, but is recognized by the human immune system and therefore has potential as a diagnostic antigen. Our results provide a mechanistic explanation as to how Histoplasma survives the encounter with phagocytes by producing Sod3 and a dual catalase system to detoxify host-derived anti-microbial reactive oxygen.