The long term goal of the proposed studies is the elucidation of the biochemical and molecular mechanisms by which Entamoeba histolytica adheres to host cells to initiate contact-dependent cytolysis. E. histolytica is an important cause of diarrhea in the Acquired Immunodeficiency Syndrome and is the third leading parasitic cause of death in the developing world. Amebic adherence to colonic mucus is the first step in the pathogenesis of invasive amebiasis and is required for E. histolytica contact-dependent lysis of host cells. The initial studies by the principal investigator identified the amebic protein which mediates adherence to Chinese hamster ovary (CHO) cells and human colonic mucus as a galactose (Gal) and N-acetyl-D-galactosamine (GalNAc)-binding lectin. The proposed studies are directed towards characterizing the role of the adherence lectin and its individual heavy and light subunits in amebic adherence and target cell cytolysis. The subunits will be purified and the ability of the isolated subunits to bind galactose or competitively inhibit amebic adherence tested. Subunit-specific monoclonal and polyclonal antibodies will be produced and used to determine the contributions of the individual subunits to amebic adherence and contact-dependent cytotoxicity. Antigenic determinants and functional domains of the adherence lectin will be defined by competitive binding of monoclonal antibodies. The adherence protein's subunits will bc fragmented chemically or enzymatically in order to map antigenic and functional domains defined by monoclonal antibodies to peptide fragments of the adherence lectin. A monoclonal antibody-based radioimmunoassay (RIA) for the adherence lectin that has been produced will be tested for its clinical utility in detecting amebic antigen in serum, urine, and liver abscess fluid from patients with amebiasis. The isolation and characterization of the adherence lectin will be central to understanding the pathogenesis of E. histolytica infection and to the possible development of a vaccine.
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