MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF A SECRETED LIPASE IN LEISHMANIA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed research is designed to contribute to the understanding of mechanisms of Leishmania survival, growth and development. The disease spectrum of leishmanias is ranges from mild self-limiting cutaneous ulcers to a potentially fatal visceral infection, each dependant on the species of parasite with which one is infected. Proteins secreted by Leishmania are of interest because it is through these molecules that the parasites are able to sense, respond to, and alter their environments. Such secreted proteins could make unique suitable targets for the development of new compounds to prevent or treat this disease. Currently there is no vaccine available for the prevention of transmission of these parasites, and toxic antimony compounds are often used unsuccessfully for its treatment. Lipases are lipolytic enzymes that hydrolyze the ester bond of triglycerides. In other systems, lipase activity is involved in the reorganization of membrane structure, cell signaling, or nutrient acquisition. These observations suggest that lipase activity is essential for Leishmania although the biological significance of this enzyme has not been proven experimentally. The working hypothesis of this study is that lipase is essential to Leishmania. The following approaches will be taken to determine the role of this enzyme in the parasite life cycle: 1) the full-length copy of the lipase gene will be identified and characterized. The expression of lipase will be examined though out the parasite developmental life cycle and the phylogenic conservation of lipase among kinetoplastid protozoa will be determined; 2) Null mutants, deficient in lipase activity, will be generated and tested for viability; 3) The lipase gene will be expressed as a recombinant protein and assayed for substrate preference. In addition, various classes of organophosphate inhibitors of lipase will be tested to determine the sensitivity of the leishmanial lipase to these compounds.