GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫糖基化突变体

• 批准号: 2886693
• 负责人: Salvatore J Turco
• 金额: $ 50.41万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2886693
• 关键词: Leishmania donovani; disaccharides; enzyme activity; flow cytometry; gene expression; gene mutation; gene targeting; genetic regulation; glycolipids; glycoprotein biosynthesis; glycosylation; hamsters; host organism interaction; intracellular parasitism; laboratory mouse; leishmaniasis; membrane proteins; molecular cloning; northern blottings; nucleic acid sequence; phosphatidylinositols; polymerase chain reaction; protein structure function; southern blotting; transposon /insertion element; virulence

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.

利什曼原虫的一个显著特征是它们的生存能力 在它们整个生命周期中遇到的恶劣环境中。 脂磷多糖(LPG)是细胞表面的主要糖偶联物。 利什曼原虫，在利什曼原虫的生存中起着关键作用 寄生虫。先前的研究已经将来自L.donovani的液化石油气定性为 [-6Gal(β1，4)MAN 1-PO4]的重复二糖单元的聚合物， 通过糖核连接到一种新型的裂解烷基磷脂酰肌醇上 抛锚。一种有效的方法建立生物合成和 LPG的功能是识别编码蛋白质的基因 它的合成所需的基因，以及有缺陷的突变体的开发 液化石油气生物合成的一个或多个方面。 我们已经开发出有效的方法来创造液化石油气突变体，并为 液化石油气生物合成基因的鉴定 这些突变体的缺陷。由此，5个不同的基因参与了液化石油气 已经鉴定了生物合成，以及它们的生化和生物学 已建立的角色。我们的研究为我们提供了对一般 与许多真核生物相关的糖共轭合成机制，以及 其中几种编码的蛋白质有望成为未来的靶标 化疗的努力。 最终目标是全面了解 参与液化石油气生物合成的基因，这种复合体的组织和 重要的生化途径，包括它的细胞学 以及液化石油气的生物功能。我们的特定 目标是： L)寻找新的液化石油气突变体和鉴定缺陷基因 多诺瓦利什曼原虫。我们还将探索一种令人兴奋的新方法， 使用寄生虫内的转座元件来标记和灭活 液化石油气基因。 2)确定每种液化石油气突变的生化后果。 液化石油气的生物合成，以及与液化石油气有关的分子， 如磷酸二糖重复单元和GPI锚点。 3)确定各自编码的蛋白质(S)的生化作用 LPG基因。 4)利用精心挑选的液化石油气突变体和对照来建立 每个基因在寄生虫感染循环中的作用。