The targets of this proposal are the functions and regulatory mechanisms of two Histoplasma capsulatum (Hc) genes that are up-regulated early in macrophage infection and may be important for pathogenic success in the hostile host environment. Histoplasmosis is the most common endemic mycoses in the world and is particularly dangerous for immunocompromised patients. Disease manifestations may be pulmonary or systemic, resulting from the respiratory route of infection and dissemination through the mononuclear phagocytic system. From host inhalation of mold elements through conversion to a budding yeast, entry in macrophages, and survival within a harsh intracellular compartment, this dimorphic fungus successfully faces a wide range of environmental stimuli and threats from host defense mechanisms. The ability for adaption to the host by a soil microorganisms is intriguing from an evolutionary standpoint and clinically significant. Examining genes that are specifically up regulated during infection can elucidate pathogenic mechanisms and the nature of the host micro-environmental niche in which the fungus persists. Such studies may also reveal new vaccine candidates or therapeutic drug targets. Differential display (dddRT-PCR) and in vivo expression technology (IVET) was used to identify a number of Hc early response genes including yps-3 and a gene encoding a small transcript in antisense orientation to a homology of an immunogenic protein found in the cell wall and culture supernatant. Its predicted homology with mammalian EGF-like proteins and a domain of the Blastomyces dermatitis WI-1 antigen is consistent with potential roles in attachment or intracellular signaling. DdRT-PCR was used identify yps-3 up regulation during infection and moreover revealed 3' untranslated region processing and alternate polyadenylation associated with novel sequence motifs.
The first aim i s to determine the function, pathogenic role and in vivo regulatory mechanisms for yps-3. IVET was used to identify up regulation during infection of the other gene targeted in this proposal.
Our second aim i s to determine the function of this gene, including its role in potential antisense down regulation of the protein kinase homolog as part of the fungus's adaption to the host intracellular environment. Both yps-3 and IVET-identified gene are up regulated within four hours after intracellular infection.
The third aim i s to determine the environmental stimuli regulating expression, using specific conditions relevant to Hc pathogenesis as well as macrophage cell culture and mouse infection models. These studies are designed to characterize unique biological aspects of each gene as well as potentially shared features of fungal adaptive responsiveness in a pathogenically relevant setting.
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