GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫糖基化突变体

基本信息

批准号：
7151926
负责人：
Stephen M Beverley
金额：
$ 68.68万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-07-01 至 2008-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7151926
关键词：
Acute Anabolism Animals Biochemical Bioinformatics Biological Models Candidate Disease Gene Carrier Proteins Class Critical Pathways Cutaneous Leishmaniasis Data Defect Disease Elements Enzymes Family Genes Genetic Genetic Models Genome Glycocalyx Glycoconjugates Goals Individual Infection Insecta Intervention Knock-out Laboratories Leishmania Leishmania major Maps Methodology Methods Modeling Molecular Molecular Chaperones Mus Mutation Organism Parasites Pathology Pathway interactions Play Property Proteins Reporting Role Sand Flies Signal Pathway Sphingolipids Structure Surface System Testing Vaccines Vertebral column Virulence Virulent Visceral Leishmaniasis base chemotherapy design genetic analysis genome sequencing glycosylation glycosyltransferase improved lipid metabolism lipophosphonoglycan macrophage mutant novel positional cloning sugar nucleotide vaccination strategy

项目摘要

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.

利什曼原虫是重要的热带原生动物寄生虫，导致超过 1000 万人患病全世界。通过了解这种寄生虫生存和引起疾病的机制，可以设计和开发改进的化疗和疫苗接种策略。这里我们重点关注的是寄生虫表面，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发挥着重要作用寄生虫生存和毒力中的作用。通过产生 LPG 突变体，然后分离拯救基因针对这一缺陷，我们之前已经建立了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，并了解特定的 LPG 基因和糖复合物如何导致疾病。这些研究将集中于大型利什曼原虫，皮肤利什曼病的病原体，因为它具有许多实验优势包括在明确的动物和昆虫模型中检查完整感染周期的能力，以及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针对 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 突变体来识别相关的 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研究中被研究过生物; 2) 使用“反向遗传”方法，结合利什曼原虫基因组计划和生物信息策略，识别候选基因，并确定它们在液化石油气生物合成中的作用； 3）创建在完全有毒的寄生虫背景下，LPG基因中的“敲除”突变体无效，包括相关对照，以及然后利用它们来绘制它们对 LPG 家族糖复合物的合成和结构的影响，以及在涉及小鼠、巨噬细胞和沙蝇感染的测试中，它们对寄生虫毒力的影响；和 4) 到剖析 LPG 关键结构域损害毒力的分子机制相关糖复合物。作为一个例子，我们将研究一种遗传模型（磷酸聚糖缺乏），该模型将急性毒力与持久性分开，并探索其在疫苗策略中的效用。