GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫糖基化突变体

• 批准号: 3146079
• 负责人: Salvatore J Turco
• 金额: $ 30.39万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3146079
• 关键词: Leishmania donovani; cell membrane; electroporation; enzyme activity; gene complementation; gene expression; gene mutation; genetic regulation; glycolipids; glycoprotein biosynthesis; glycosylation; host organism interaction; intracellular parasitism; leishmaniasis; membrane proteins; northern blottings; nucleic acid sequence; polymerase chain reaction; pulsed field gel electrophoresis; restriction mapping; southern blotting; transfection; virulence

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.

利什曼原虫的一个显著特征是它们能够在 在其整个生命周期中遇到的水解性环境。 脂磷多糖(LPG)是细胞表面的主要糖偶联物。 利什曼原虫的前鞭毛虫，据信在 寄生虫在恶劣环境中的生存。我们已经刻画了 来自杜诺瓦尼乳杆菌的液化石油气是一种重复的双糖单元的聚合物 PO4-&gt；6Gal(β1，4)人通过磷酸糖核连接到一种新的 赖素烷基磷脂酰肌醇锚定。其中一个强大的方法是 建立液化石油气的生物合成和功能是分离， 牛血清白蛋白糖基化变异体的特性及其利用 表达缺陷液化石油气的寄生虫。这种方法的成功和 遗传方法的快速发展，如DNA 利什曼原虫中的转基因，需要一种全面的遗传方法来 对这种重要寄生虫毒力决定因素的研究。这些 研究应该使我们第一次能够分离出特定的基因 参与液化石油气的生物合成，并探讨其遗传组织 寄生虫基因组中负责液化石油气生物合成的部分。这个 本申请的具体目标如下： 1.分离杜氏乳杆菌糖基化突变体 缺乏LPG或表达缺陷LPG分子的前鞭毛体 它们的细胞表面。至少25-30个LPG中有缺陷的独立克隆 生物合成是可能的。 2.LPG突变体的生化和免疫学分类。由In 体外糖基化试验，我们将确定酶缺陷在 导致突变的LPG生物合成途径。 3.LPG-L.donovani突变基因的功能遗传学分离 互补性。 4.LPG+互补宇宙的遗传特征。