Project Summary Histoplasma capsulatum is one of several systemic dimorphic fungal pathogens that switch their growth program from an infectious mold form in the soil to a pathogenic yeast form in mammalian hosts. H. capsulatum causes up to 25,000 life-threatening infections per year in the U.S. alone with up to 50% mortality rate, and is the most common cause of fungal respiratory infections in healthy hosts. Infection occurs when the soil is disrupted, facilitating dispersion of hyphal fragments or spores that are inhaled by humans. Spores and hyphal fragments are the primary infectious agents; however, once introduced into the host, the pathogen converts to a budding-yeast form, which survives and replicates within host macrophages. In the laboratory, the switch between the infectious and parasitic states is modeled by changing the growth temperature: cells grow in the filamentous form (hyphal) at room temperature, whereas growth at 37ºC is sufficient to trigger growth in the yeast form and expression of virulence factors. Despite its importance to human health, very little is known about how H. capsulatum senses and responds to human body temperature. Our prior research findings significantly contributed to the understanding of the molecular mechanism used by H. capsulatum to regulate cell morphology and virulence gene expression: we found that four transcriptional regulators, Ryp1,2,3,4, are the core components of a temperature-responsive intersecting regulatory network. In unpublished studies, we comprehensively identified Ryp-interacting proteins with potential regulatory roles. Among the diverse set of Ryp2-interacting proteins, we characterized a heat shock protein, Hsp90, and two proteins, Ssk1 and Skn7, with predicted response regulator domains. We found that Hsp90, Ssk1 and Skn7 regulate yeast phase growth in H. capsulatum. Hsp90 plays a key role in the heat shock response; and response regulators work with sensor histidine kinases and are often involved in sensing environmental signals. In this project, we propose to build upon our previous findings and fully characterize the involvement of the heat shock response and histidine kinase pathways in regulating Ryp proteins, cell morphology and virulence traits in H. capsulatum in response to host temperature. These studies will provide fundamental information on how cells sense temperature and turn on the appropriate virulence pathways in the host. Ultimately, the information obtained from this project can be used to develop therapeutics for H. capsulatum infections and help prevent other dimorphic fungal infections.