Entamoeba histolytica is a human cytolytic parasite with worldwide distribution that infects up to 50 million people and results in an estimated 70,000 deaths annually. The E. histolytica Ga1NAc lectin mediates trophozoite adherence and plays a role in contact-dependent killing of human immune effector and epithelial cells. The cytolytic capacity of E. histolytica is central to pathogenesis yet little is known about how cytolysis is initiated after cell contact via the Ga1NAc lectin. The hypothesis of this proposal is that protein tyrosine kinases and/or their substrates are intimately involved in the signal transduction by the Ga1NAc lectin. Protein tyrosine kinases (PTK) and/or their substrates have been demonstrated by co-immunoprecipitation to associate with the Ga1NAc lectin. We propose to test the role of these PTK/substrates in lectin- mediated signal transduction. Ga1NAc lectin-associated PTK/substrates will be identified from lectin co-immunoprecipitations or by the yeast two hybrid system (using the lectin cytoplasmic domain as bait). Lectin-associated PTK/substrates will be cloned and sequenced, the primary amino acid sequence characterized, and mAb produced against them. These reagents will be used to assess changes in the PTK/substrate activity, phosphorylation state and intracellular location during signaling. Finally genetic characterization will be attempted using reverse genetic approaches. Successful completion of these experiments will provide insight into the role and identity of PTK/substrates in lectin-mediated killing and may establish the basis for novel therapeutic or prophylactic measures against amebiasis.
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