Invasion of host tissue by helminth larvae is the first step in infection of the human host by several nematode and trematode parasites. Previous work has shown that a key factor in this process is the release of tissue- degradative proteolytic enzymes by the infectious larvae. Using schistosome cercariae as a model system, this step in host infection will be dissected at the molecular level. Taking advantage of a large schistosome laboratory life cycle, purification protocol for the target enzyme, cDNA and genomic clones, a high level recombinant expression system, and a computer model of the three-dimensional structure of the active site of the cercarial proteinase, the following questions will be addressed: What is the molecular basis for the broad substrate activity of the proteinase which makes it so well suited to facilitate cercarial invasion? How does the parasite protect itself from the action of its own enzyme? What are the steps involved in biosynthesis, packaging and release of the proteinase during cercarial development and host invasion? What unique molecular characteristics of cercarial proteinase resulted from its molecular evolution and underlie its function and specificity? Does the proteinase also play a role in transformation of cercaria to schistosomulum? The specific studies to address these questions include the following: (i) Analysis of active site substrate specificity of the cercarial proteinase using synthetic peptide substrates and inhibitors. (2) Confirmation of key residues identified by computer modeling of the active site using site-directed mutagenesis. (3) X-ray crystallographic studies of the three-dimensional structure of the enzyme. (4) Identification of the site of zymogen activation. (5) Construction of phylogenetic trees of the molecular evolution of the proteinase. Direct tests of the hypotheses that the cercarial proteinase facilitates cercarial invasion, and that it is required for cercaria-to-schistosomulum transformation, will be carried out by observing the effects of specific synthetic inhibitors in an in vitro model of human skin invasion and transformation, and analysis of protection against cercarial invasion by induction of an immune response against the proteinase in a murine model of infection.
