A striking feature of Leishmania parasites is their ability to survive in hostile environments encountered throughout their life cycle. Lipophosphoglycan (LPG) is the major cell surface glycoconjugate of all Leishmania promastigotes and plays a key role in the survival of the parasites. Prior studies have characterized the LPG from L. donovani as a polymer of repeating disaccharide units of [-6Gal(beta1,4)Man 1-PO4], attached via a glycan core to a novel lyso-alkylphosphatidyl inositol anchor. One powerful approach for establishing the biosynthesis and function of LPG is the identification of genes encoding proteins required for its synthesis, and the development of mutants defective in one or more aspects of LPG biosynthesis. We have developed effective methods for creating lpg- mutants, and for the identification of LPG biosynthetic genes capable of rescuing the defect in these mutants. With these, 5 different genes involved in LPG biosynthesis have been identified, and their biochemical and biological roles established. Our studies have provided new insight into general mechanisms of glycoconjugate synthesis relevant to many eukaryotes, and several of the encoded proteins show promise as targets for future chemotherapeutic efforts. The ultimate goal is to develop an comprehensive understanding of the genes involved in LPG biosynthesis, the organization of this complex and important biochemical pathway including its cellular compartmentalization, and the biological function of LPG.
Our specific aims are: l) To identify new lpg- mutants and identify the defective genes in Leishmania donovani. We will also explore an exciting new approach, using transposable elements within the parasite, to tag and inactivate LPG genes. 2) To determine the biochemical consequences of each lpg- mutation on the biosynthesis of LPG, and molecules bearing LPG-related elements, such as the disaccharide phosphate repeating unit and GPI anchors. 3) To determine the biochemical role of the protein(s) encoded by each LPG gene. 4) To utilize carefully chosen lpg- mutants and controls to establish the role of each gene in the parasite infectious cycle.
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