GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫糖基化突变体

• 批准号: 2423984
• 负责人: Salvatore J Turco
• 金额: $ 50.67万
• 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/2423984
• 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.

利什曼原虫寄生虫的一个显着特征是它们的生存能力 在其整个生命周期中遇到的恶劣环境中。 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在生存中发挥着关键作用 寄生虫。先前的研究已将杜氏乳杆菌的液化石油气描述为 [-6Gal(beta1,4)Man 1-PO4]重复二糖单元的聚合物， 通过聚糖核心连接到新型溶血烷基磷脂酰肌醇 锚。一种建立生物合成和 LPG 的功能是识别编码蛋白质的基因 其合成所需，以及缺陷突变体的开发 LPG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建LPG突变体，并用于 鉴定能够拯救液化石油气生物合成基因 这些突变体的缺陷。有了这些，LPG 涉及 5 个不同的基因 生物合成已被鉴定，其生化和生物 角色已建立。我们的研究为一般性问题提供了新的见解 与许多真核生物相关的糖复合物合成机制，以及 几种编码的蛋白质有望成为未来的目标 化疗努力。 最终目标是全面了解 参与液化石油气生物合成的基因，该复合体的组织和 重要的生化途径，包括其细胞 区室化和液化石油气的生物学功能。我们的具体 目标是： l) 鉴定新的lpg-突变体并鉴定其中的缺陷基因 杜氏利什曼原虫。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) 确定每个lpg-突变对生化的影响 液化石油气的生物合成以及带有液化石油气相关元素的分子， 例如二糖磷酸重复单元和 GPI 锚。 3) 确定每个编码的蛋白质的生化作用 液化石油气基因。 4) 利用精心选择的lpg-突变体和对照来建立 每个基因在寄生虫感染周期中的作用。