Molecular and Biochemical Characterization of Lipases Secreted by Leishmania (2009 - Present)

Investigator:  Alison Shakarian, Salve Regina University 
Mentor: Dennis Dwyer, LPD/NIAIS

Abstract:  The proposed research is designed to contribute to the understanding of mechanisms of Leishmania survival, growth and development. Several species of Leishmania are pathogens of humans. At least 12 million people in Africa, India and Latin America are infected with Leishmania, and 350 million are at risk. Moreover, the thousands of US troops currently deployed in endemic areas (i.e. the Middle East) are at significant risk for contracting the parasite and significant disease. 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 group of diseases as currently there is no vaccine available against these parasites and toxic pentavalent 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 and in some cases for nutrient acquisition. This suggests that lipase is an essential enzyme for Leishmania although presently its biological significance has not been proven experimentally. A characterization of leishmanial lipase could lead to a better understanding of these mechanisms in kinetoplastid protozoa. The working hypothesis, that lipase is essential to Leishmania biology/survival, will be tested in this proposal. The following approaches to determine the role of this enzyme in the parasite life cycle are planned:  1) To continue characterization of the lipase gene at the molecular level, using sequence and Southern blot analyses; the expression of lipase will be examined though out the parasite developmental life cycle using real-time RT-PCR; and the 2) the phylogenetic conservation of lipase among kinetoplastid protozoa will be determined by pulse-field gel electrophoresis, sequence analysis and enzyme assays. 3) To characterize the leishmanial lipase enzyme activity and determine its sensitivity to organophosphate inhibitors, the lipase gene will be expressed and assayed for substrate preference. Finally, sensitivity of this protein to organophosphate inhibitors will be tested.