The overall goal of this project is to further our understanding of the molecular basis of host-parasite interactions of Cryptosporidium parvum in order to develop specific preventive and therapeutic strategies for cryptosporidiosis. C. parvum is a significant cause of human gastrointestinal disease worldwide. In immunocompromised hosts, such as AIDS patients, C. parvum may cause severe, protracted and ultimately fatal disease. There is currently no effective specific therapy for the disease caused by this parasite. This proposal is focused on elucidating the molecular basis for the interaction of gp40 and gp 15 with host cells and on characterization of the post-translational processing and modifications of gp40/15 and its products, which may facilitate the identification of novel biochemical targets for future drug discovery. These glycoproteins are likely to be involved in attachment and invasion since they are present on the surface of invasive stages of the parasite, bind to host cells, competitively inhibit invasion, and are targets of infection-neutralizing antibodies and lectins. They are derived by proteolytic cleavage of a precursor protein gp40/15 which is encoded by a single copy gene, Cpgp40/15. This gene is highly polymorphic, particularly in human (genotype I) isolates. In the first Specific Aim, the role of these proteins in mediating attachment and invasion will be examined in detail. Binding of native C parvum gp40 and gpl5 as well as recombinant gp40 and gpl5 (derived from isolates of both genotype) to host cells will be assessed. The ability of the recombinant proteins to inhibit infection in vitro will be determined. The structural basis of attachment of gp40 and gpl5 to host cells will be determined by site-directed mutagenesis. Host cell receptors for gp40 and gpl5 will be identified. In the second Specific Aim, post-translational processing of the precursor protein gp40/15 will be investigated and studies on the protease involved in processing initiated. Glycosylation of these glycoproteins will be examined. The long-term objective is to determine if infection by this parasite can be prevented or treated by targeting molecules such as these glycoproteins as well as enzymes involved in their post-translational processing and modifications.
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