GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫糖基化突变体

• 批准号: 3146080
• 负责人: Salvatore J Turco
• 金额: $ 33.44万
• 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/3146080
• 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 在其生命周期中遇到的水解环境。 脂磷酸聚糖（LPG）是所有细胞的主要细胞表面糖缀合物。 利什曼原虫前鞭毛体，并被认为发挥了关键作用， 寄生虫在恶劣环境中的生存。 我们有特点 L. Donovani作为重复二糖单元的聚合物， PO 4-> 6 Gal（beta1，4）Man通过磷酸糖核心连接到新的 溶血烷基磷脂酰肌醇锚。 一种强大的方法， 建立LPG的生物合成和功能是分离， 的糖基化变体的鉴定和利用。 表达有缺陷LPG的寄生虫 这种方法的成功， 遗传学方法的快速发展，如DNA 转染利什曼原虫，保证了一个全面的遗传方法， 这一重要寄生虫毒力决定因子的研究。 这些 这些研究将使我们能够首次分离出特定的基因， 参与LPG生物合成，并探索其遗传组织， 负责LPG生物合成的寄生虫基因组部分。 的 这项申请的具体目标如下： 1.分离L.杜氏 前鞭毛体缺乏LPG或表达有缺陷的LPG分子， 它们的细胞表面 至少25-30个独立克隆在LPG中有缺陷 生物合成是可能的。 2. LPG-突变体的生化和免疫学分类。 采用 体外糖基化测定，我们将建立酶的缺陷， LPG生物合成途径解释了它们的突变。 3.分离LPG-L中突变的基因。Donovani功能遗传学 互补 4. LPG+补充Cosmetic的遗传特征。