LEISHMANIA-MACROPHAGE CELLULAR INTERACTIONS IN VITRO

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Our long term objectives are to elucidate the questions of how Leishmania infect macrophages, and how these parasites subsequently differentiate, survive and multiply in these phagocytes. Understanding the biochemical and molecular mechanisms of such host-parasite cellular interactions will provide leads for developing more effective chemo and/or immuno- therapy and -prophylaxis for leishmaniasis. A Leishmania major surface glycoprotein (gp63) was purified from promastigotes and found to be a metallo-protease active at acidic pH. It appears to mediate the binding of Leishmania to macrophages and to protect parasites from degradation in phagolysosomes. Leishmania virulent phenotype is associated with the abundance of gp63 and tunicamycin-resistance, marked by DNA amplification and an increased level of tunicamycin-sensitive N- acetylglucosamine-1-phosphate transferase (NAGT) in the dolichol pathway. This is a continuation of above-mentioned work to study cell biology, biochemistry and molecular biology of gp63 as a virulent determinant and the regulation of its function by N-glycosylation in Leishmania mexicana spp. as follows: (1) Further characterization of gp63 as a metallo-protease for developing specific inhibitors; (2) Purification of gp63 from amastigotes for studying its protease activity; (3) Understanding the protective functions of gp63 in phagolysosomes by studying interactions of macrophages with gp63-coated liposomes; (4) Analyses of intracellular routing of gp63, the structure of its glycans and Leishmania NAGT to understand the regulatory role of N-glycosylation in the function of gp63 as a virulent determinant; (5) Characterization of NAGT and other potential virulent genes in amplified DNA of tunicamycin-resistant cells by molecular cloning in expression vectors. The results of these studies will help not only our understanding leishmanial virulence in intracellular parasitism of macrophages but also metallo-protease, glycoprotein and dolichol pathway in general.