LEISHMANIA-MACROPHAGE CELLULAR INTERACTIONS IN VITRO

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Our long term objectives are to elucidate the questions of how leishmanias infect macrophages, and how these parasites subsequently differentiate, survive and multiply in these phagocytes. Understanding the cellular and molecular mechanisms of such host-parasite interactions will provide leads for developing more effective chemo- and/or immunotherapy and prophylaxes for leishmaniasis. Experiments proposed here represent a continuation of previous work using Leishmania mexicana amazonensis-J774 G8 macrophage in vitro system to study leishmania-macrophage membrane interactions and regulatory mechanism of membrane protein biosynthesis during leishmanial differentiation in macrophages. We have previously prepared a panel of monoclonal antibodies specific to surface molecules of the parasite. One particular monoclonal, 6H12, recognizes a major surface antigen consisting of glycopeptide subunits (65-68 kilodalton) after SDS-PAGE. The membrane antigen appears to play a role in the survival of leishmanias in the macrophages. In other experiments, we have found that the regulatory mechanism of protein biosynthesis during leishmanial differentiation is different even for closely related proteins, i.e., alpha- and beta-tubulins. On the basis of the above findings, we now propose to: 1) attempt to purify the major surface antigen using monoclonal antibody affinity chromatography; 2) probe the function of the antigen(s) in their interactions with macrophages and hydrolytic enzymes after deglycosylation, using, for example, tunicamycin and monoclonal antibody-mediated modulation of leishmania membrane glycoprotein(s); 3) study the biosynthesis, expression, processing and degradation of the major surface antigen, especially during leishmanial differentiation in macrophages; 4) study differentiation-related regulatory mechanisms of leishmanial membrane glycoprotein synthesis in in vitro protein translation sytem using mRNAs. The specificity of the monoclonal antibodies already prepared makes it possible to identify and to quantitate particular antigen(s) in these proposed experiments. The results of these studies will help not only our understanding the biochemistry, cell and molecular biology of leishmanial membrane glycoproteins, but also their roles in host-parasite interactions in leishmaniasis.