Amebiasis, which results from tissue invasion by the protozoan parasite, Entamoeba histolytica, is a major cause of mortality and morbidity in impoverished countries and is of significance in the United States. The molecular mechanism of interaction of E. histolytica with mammalian target cells is poorly understood. Contact of the parasite with a target cell and action of the amoeba actin cytoskeleton are required. There is evidence that parasite attack is receptor mediated, that the receptor recognizes target carbohydrates, and that lipid components of the target cell membrane trigger the attack response. The long term goals of this research are to contribute to the explanation of the mechanism of interaction of E. histolytica with mammalian cells and to identify molecules which may be exploited to develop immunological or other clinical means of prevention of Amebiasis.
The specific aims of this proposal are to: identify target cell membrane lipid ligands which trigger amoeba actin polymerization, a signal of the initiation of attack; prepare monoclonal anti-amoeba antibodies which inhibit this attack response; use the monoclonal antibodies to aid isolation and characterization of amoeba antigens involved in attack; use attack stimulating liposomes to seek evidence of biochemical controls in the attack mechanism, specifically, changes in intracellular calcium flux and protein phosphorylation in response to ligand-receptor interaction. Liposomes will be constructed with purified human red blood cell glycosphingolipids (collaboration with Dr. Sen-Itiroh Hakomori) and tested for their ability to stimulate E. histolytica actin polymerization as detected by enhanced rhodamine-phalloidin binding. Monoclonal antibodies against E. histolytica membranes will be detected by their ability to inhibit the amoeba cytoskeleton response. Immunoaffinity techniques will provide the basis for identification and isolation of specific attack related amoeba antigens. Changes in intracellular calcium triggered by target ligand bearing liposomes will be detected by fluorescence microscopy and video image analysis using the calcium sensitive fluor, Fura-2. Liposome stimulated changes in protein phosphorylation will be detected by 2-dimensional electrophoresis and autoradiography of 32P-labeled E. histolytica proteins.
