The initial step in the infection of the human host by schistosome parasites is invasion of skin by an aquatic larval form, the cercaria. We have been studying the molecular mechanisms by which the cercaria can invade skin and ultimately gain access to the bloodstream. Our proposed work builds upon previous studies in which we isolated and characterized a secreted serine protease that appears to facilitate cercarial invasion by degradation of the macromolecular components of epidermis and dermis. To learn more about how this enzyme specifically functions to degrade host macromolecules, we have produced a three dimensional computer model of its active site. This model will first be tested by assay of specific peptide substrates and inhibitors predicted by the model to be effective, as well as by activity and thermal stability assays following site-directed mutagenesis of recombinant enzyme. The model can then be used to identify potentially important regions of substrate binding relevant to cleavage of host substrates like elastin. We will also test the effective of specific proteinase inhibitors, predicted by the model to be most effective, on invasion of human skin by live cercariae in vitro. In related studies, we have developed a powerful new technique that allows rapid isolation and sequencing of any eukaryotic serine or thiol proteinase gene. We will use this technique to isolate and sequence the corresponding cercarial proteinase genes from S. japonicum and S. douthitti. This will allow us to study interspecies divergence of the cercarial proteinase during schistosome evolution, and correlate any differences in substrate specificity of these enzymes with differences in protein structure.
