GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫糖基化突变体

• 批准号: 2066089
• 负责人: Salvatore J Turco
• 金额: $ 36.55万
• 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/2066089
• 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.

利什曼原虫寄生虫的一个显着特征是它们能够在环境中生存 在其整个生命周期中遇到的水解环境。 脂磷酸聚糖 (LPG) 是所有细胞表面的主要糖复合物 利什曼原虫前鞭毛体被认为在 寄生虫在恶劣环境中的生存。 我们已经表征了 来自杜诺瓦尼乳杆菌的 LPG 作为重复二糖单元的聚合物 PO4->6Gal(beta1,4)Man 通过磷酸糖核心连接到新型 溶血烷基磷脂酰肌醇锚。 一种强有力的方法 建立液化石油气的生物合成和功能是分离， 糖基化变体的表征和利用 表达有缺陷的液化石油气的寄生虫。 这种方法的成功和 DNA 等遗传方法的快速发展 利什曼原虫转染，需要采用全面的遗传方法 对这种重要的寄生虫毒力决定因素的研究。 这些 研究应该使我们能够首次分离出特定的基因 参与液化石油气的生物合成，并探究其遗传组织 负责 LPG 生物合成的寄生虫基因组部分。 这 该应用的具体目标如下： 1. 分离杜氏乳杆菌的额外糖基化突变体 缺乏 LPG 或表达有缺陷的 LPG 分子的前鞭毛体 他们的细胞表面。 至少 25-30 个独立克隆存在 LPG 缺陷 生物合成是可能的。 2. LPG-突变体的生化和免疫学分类。 由在 体外糖基化测定，我们将确定酶促缺陷 LPG生物合成途径导致其突变。 3. 通过功能遗传学分离 LPG-杜氏乳杆菌中突变的基因 互补。 4. LPG+互补粘粒的遗传特征。