GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫糖基化突变体

• 批准号: 6579518
• 负责人: Stephen M Beverley
• 金额: $ 66.06万
• 依托单位: WASHINGTON UNIVERSITY
• 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 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6579518
• 关键词: Leishmania major; gene expression; genetic mapping; genetically modified animals; glycolipids; glycoproteins; glycosylation; host organism interaction; membrane activity; pathologic process; phosphoglycerate; virulence

DESCRIPTION (provided by the applicant): Leishmania are important tropical protozoan parasites, causing disease in more than 10 million people worldwide. By understanding the mechanisms used by this parasite to survive and cause disease, it will be possible to design and develop improved chemotherapy and vaccination strategies. Here we focus on the parasite surface, which is covered by a dense glycocalyx consisting primarily of lipophosphoglycan (LPG). This GPI-anchored polyphosphosaccharide, along with structurally related glycoconjugates, play important roles in parasite survival and virulence. By generating LPG mutants and then isolating genes which rescue this defect, we have previously established a powerful genetic system to probe parasite virulence. We will use this methodology to develop a comprehensive understanding of the LPG biosynthetic pathway, and to understand how specific LPG genes and glycoconjugates contribute to disease. These studies will focus on Leishmania major, the agent of cutaneous leishmaniasis, as it offers many experimental advantages including the ability to examine the complete infectious cycle in well defined animal and insect models, and soon the complete genome sequence will be available. Preliminary data from our laboratory suggest that these studies of L. major LPG are applicable to L. donovani as well, the agent of fatal visceral leishmaniasis. Our specific aims are 1) to use LPG mutants in a forward genetic approach to identify the relevant LPG biosynthetic genes; typically these are 'new' genes whose activity has not been studied previously in any organism; 2) to use 'reverse genetic' approaches, in combination with the Leishmania genome project and bioinformatic strategies, to identify candidate genes, and establish their role in LPG biosynthesis; 3) to create null 'knockout' mutants in LPG genes in a fully virulent parasite background, including relevant controls, and then to use these to map out their effects on the synthesis and structure of LPG family glycoconjugates, and their effect on parasite virulence in tests involving infections of mice, macrophages and sand flies; and 4) to dissect the molecular mechanisms by which virulence is compromised by key structural domains of LPG and related glycoconjugates. As one example, we will study a genetic model (phosphoglycan-deficiency) which separates acute virulence from persistence, and explore its utility in vaccine strategies.

描述（由申请人提供）：利什曼原虫是重要的热带原生动物寄生虫，导致全世界超过 1000 万人患病。通过了解这种寄生虫生存和引起疾病的机制，将有可能设计和开发改进的化疗和疫苗接种策略。在这里，我们重点关注寄生虫表面，该表面被主要由脂磷酸聚糖 (LPG) 组成的致密糖萼覆盖。这种 GPI 锚定的多磷酸糖以及结构相关的糖复合物在寄生虫的生存和毒力中发挥着重要作用。通过产生 LPG 突变体，然后分离挽救这一缺陷的基因，我们之前已经建立了一个强大的遗传系统来探测寄生虫的毒力。我们将使用这种方法来全面了解 LPG 生物合成途径，并了解特定 LPG 基因和糖复合物如何导致疾病。这些研究将集中于皮肤利什曼病的病原体——大型利什曼原虫，因为它具有许多实验优势，包括能够在明确的动物和昆虫模型中检查完整的感染周期，并且很快就会获得完整的基因组序列。我们实验室的初步数据表明，这些对 L. Major LPG 的研究也适用于致命性内脏利什曼病的病原体杜诺瓦尼利什曼氏菌。 我们的具体目标是1）在正向遗传方法中使用LPG突变体来鉴定相关LPG生物合成基因；通常，这些是“新”基因，其活性以前从未在任何生物体中进行过研究； 2）利用“反向遗传”方法，结合利什曼原虫基因组计划和生物信息学策略，鉴定候选基因，并确定其在LPG生物合成中的作用； 3) 在完全毒力的寄生虫背景（包括相关对照）中在 LPG 基因中创建无效的“敲除”突变体，然后使用这些突变体来绘制它们对 LPG 家族糖复合物的合成和结构的影响，以及它们对寄生虫毒力的影响涉及小鼠、巨噬细胞和沙蝇感染的测试； 4) 剖析 LPG 和相关糖复合物的关键结构域损害毒力的分子机制。作为一个例子，我们将研究一种将急性毒力与持久性分开的遗传模型（磷酸聚糖缺乏），并探索其在疫苗策略中的效用。