Invasive amoebiasis is a major health problem in developing countries and within certain segments of the U.S. population. It is characterized by the massive destruction of host tissues by the parasite Entamoeba hystolytica. It appears that simple phagocytosis is preceded by a contact-dependent cytolethal stage which may involve the transfer of a membrane active toxin from the amoeba to its target cell. This toxin produces large membrane pores, depolarizing and killing the cell. The purpose of this proposed research is to characterize the action of this material in model membranes and cells, to probe the details of the contact-killing process, and, thereby, to establish whether this material is indeed a central factor underlying the disease. The material will be purified and antibodies grown to it. The purified material will be tested for pore-forming activity in lipid vesicles using a newly developed method employing voltage sensitive fluorescent dyes. This will allow determination of the rate of insertion of the pore into the membrane and provide an assay for pore-forming activity as a function of the membrane lipid composition, the presence of antibody, and the virulence of the strain of amoeba from which the toxin was isolated. The effect of the toxin on a variety of cells will be similarly assessed using a series of newly proposed voltage sensitive fluorescent probes which are designed to be used in microfluorometry. Finally, these results can be compared with those obtained from microfluorometric investigations of the contact-killing process itself. In addition to following membrane potential in these experiments, the transfer and interaction of membrane components will be monitored. The methods developed to elucidate the details of contact-dependent cytolethal effects should be generally applicable to a variety of problems in cell biology.
