LEISHMANIA-MACROPHAGE CELLULAR INTERACTIONS IN VITRO

利什曼原虫-巨噬细胞体外相互作用

• 批准号: 3130194
• 负责人: Kwang Poo Chang
• 金额: $ 22.71万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3130194
• 关键词: Leishmania; chromatography; dolichol; electron microscopy; electrophoresis; genetic manipulation; genetic translation; glycoproteins; glycosylation; glycosyltransferase; hamsters; host organism interaction; laboratory mouse; leishmaniasis; liposomes; lysosomes; macrophage; messenger RNA; microorganism culture; microorganism immunology; molecular cloning; natural gene amplification; peptidases; phagocytes; protein purification; protein transport; protozoal antigen; tissue /cell culture; transposon /insertion element; virulence

Our long term objectives are to elucidate the questions of how Leishmania infect macrophages, and how these parasites subsequently differentiate, survive and multiply in these phagocytes. Understanding the biochemical and molecular mechanisms of such host-parasite cellular interactions will provide leads for developing more effective chemo and/or immuno- therapy and -prophylaxis for leishmaniasis. A Leishmania major surface glycoprotein (gp63) was purified from promastigotes and found to be a metallo-protease active at acidic pH. It appears to mediate the binding of Leishmania to macrophages and to protect parasites from degradation in phagolysosomes. Leishmania virulent phenotype is associated with the abundance of gp63 and tunicamycin-resistance, marked by DNA amplification and an increased level of tunicamycin-sensitive N- acetylglucosamine-1-phosphate transferase (NAGT) in the dolichol pathway. This is a continuation of above-mentioned work to study cell biology, biochemistry and molecular biology of gp63 as a virulent determinant and the regulation of its function by N-glycosylation in Leishmania mexicana spp. as follows: (1) Further characterization of gp63 as a metallo-protease for developing specific inhibitors; (2) Purification of gp63 from amastigotes for studying its protease activity; (3) Understanding the protective functions of gp63 in phagolysosomes by studying interactions of macrophages with gp63-coated liposomes; (4) Analyses of intracellular routing of gp63, the structure of its glycans and Leishmania NAGT to understand the regulatory role of N-glycosylation in the function of gp63 as a virulent determinant; (5) Characterization of NAGT and other potential virulent genes in amplified DNA of tunicamycin-resistant cells by molecular cloning in expression vectors. The results of these studies will help not only our understanding leishmanial virulence in intracellular parasitism of macrophages but also metallo-protease, glycoprotein and dolichol pathway in general.

我们的长期目标是阐明如何 利什曼原虫感染巨噬细胞，以及这些寄生虫如何感染巨噬细胞 随后在这些中分化、生存和繁殖 吞噬细胞。 了解生化和分子 这种宿主-寄生虫细胞相互作用的机制将 为开发更有效的化疗和/或免疫提供线索 利什曼病的治疗和预防。 利什曼原虫主要表面糖蛋白 (gp63) 纯化自 前鞭毛体，被发现是一种在酸性条件下有活性的金属蛋白酶 酸碱度。 它似乎介导利什曼原虫与 巨噬细胞并保护寄生虫免于降解 吞噬溶酶体。 利什曼原虫毒力表型与 DNA 标记的 gp63 和衣霉素抗性丰度 扩增和衣霉素敏感 N- 水平增加 多醇中的乙酰氨基葡萄糖-1-磷酸转移酶 (NAGT) 途径。 这是上述细胞研究工作的延续 gp63 作为毒力的生物学、生物化学和分子生物学 决定簇及其通过N-糖基化对其功能的调节 墨西哥利什曼原虫。如下： (1)进一步 gp63 作为金属蛋白酶的表征用于开发 特异性抑制剂； (2)从无鞭毛体中纯化gp63 用于研究其蛋白酶活性； (3) 理解 研究gp63在吞噬溶酶体中的保护功能 巨噬细胞与 gp63 包被的脂质体的相互作用； (4) gp63的细胞内路由分析及其结构 聚糖和利什曼原虫 NAGT 了解其调节作用 gp63 毒力功能中的 N-糖基化 行列式； (5) NAGT和其他潜力的表征 衣霉素抗性细胞扩增 DNA 中的毒力基因 表达载体中的分子克隆。 这些研究的结果不仅有助于我们理解 巨噬细胞细胞内寄生的利什曼原虫毒力 还有金属蛋白酶、糖蛋白和多醇途径 一般的。