A striking feature of Leishmania parasites is their ability to survive in hydrolytic environments encountered throughout their life cycle. Lipophosphoglycan (LPG) is the major cell surface glycoconjugate of all Leishmania promastigotes and is believed to play a key role in the survival of the parasites in hostile environments. We have characterized the LPG from L. donovani as a polymer of repeating disaccharide units of PO4->6Gal(beta1,4)Man attached via a phosphosaccharide core to a novel lyso-alkylphosphatidyl inositol anchor. One powerful approach in establishing the biosynthesis and function of LPG is the isolation, characterization, and utilization of glycosylation variants of the parasites that express defective LPG. The success of this approach and the rapid progress in the development of genetic methods, such as DNA transfection in Leishmania, warrants a comprehensive genetic approach to the study of this important parasite virulence determinant. These studies should allow us to isolate, for the first time, specific genes involved in LPG biosynthesis, and probe the genetic organization of that portion of the parasite genome responsible for LPG biosynthesis.
The specific aims for this application are as follows: 1. To isolate additional glycosylation mutants of L. donovani promastigotes that either lack LPG or express defective LPG molecules on their cell surfaces. At least 25-30 independent clones defective in LPG biosynthesis are possible. 2. Biochemical and immunological classification of LPG- mutants. By in vitro glycosylation assays, we will establish the enzymatic defect in the LPG biosynthetic pathway accounting for their mutation. 3. Isolation of genes mutated in LPG- L. donovani by functional genetic complementation. 4. Genetic characterization of LPG+ complementing cosmids.
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