A key event in host invasion by several nematode and trematode parasites is the release of tissue-degrading proteolytic enzymes. Using the schistosome cercaria as a model system, we are characterizing this phenomenon at the biochemical and molecular level. In response to skin lipid, cercariae secrete two major proteases cercarial elastase and cercarial tryptase. We will complete mapping of the binding site specificity of the cercarial elastase by analysis of protease binding to recombinant variants of the natural protein inhibitors, turkey ovomucoid third domain, eglin C and ecotin. We identified the amino acid sequence around cleavage sites generated from the action of purified cercarial elastase on host macromolecular substrates like elastin, type IV collagen, and fibronectin. The cercarial elastase is also of interest as one of the first schistosome proteins released into the host during infection. Pilot studies suggest that vaccination with this antigen may induce a cercarial dermatitis in response to a challenge infection, an observation we will confirm and optimize by exploring variations in vaccination protocol. We also completed analysis of the active site binding specificity of the recently discovered tryptase using the strategy which was successful for the elastase studies. Antisera will be raised against purified cercarial tryptase, and a combination of immunohistochemistry, Northern blot analysis and in situ hybridization will be used to analyze regulation of the protease and specifically determine when it is synthesized during cercarial development, and where it is localized in the parasite. Finally, we have begun work establishing the first schistosome transfection system utilizing plasmid and retroviral constructs in which a reporter gene is driven by the cercarial elastase promoter following transfection into sporocyst cells. Our ultimate goal is transient transfection and expression of protease inhibitor genes (OMTKY3, eglin C or ecotin) to provide a complementary approach for exploring the biologic function of schistosome proteases. Computer Graphics Laboratory resources are used for molecular modeling.
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