Glycosylation Mutants of Leishmania

利什曼原虫糖基化突变体

• 批准号: 8279164
• 负责人: Stephen M Beverley
• 金额: $ 74.48万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8279164
• 关键词: Anabolism; Animals; Area; Bacteria; Biochemical; Biological Assay; Bite; Blood; Carrier Proteins; Collaborations; Complement; Cutaneous; Data; Deposition; Development; Disease; Enzymes; Eukaryota; Exhibits; Family; Foundations; Future; Galactosyltransferases; Gastrointestinal tract structure; Gene Family; Generations; Genes; Genetic; Genome; Glycoconjugates; Glycosyltransferase Gene; Goals; Grant; Health; Host Defense; Human; In Vitro; Individual; Infection; Ingestion; Intervention; Knowledge; Label; Leishmania; Leishmania major; Leishmaniasis; Lesion; Life; Longitudinal Studies; Membrane Microdomains; Methodology; Methods; Military Personnel; Molecular Chaperones; Mus; Parasites; Pathology; Pathway interactions; Play; Property; Proteins; Prunella vulgaris; RNA Interference; Research; Resistance; Risk; Role; Sand Flies; Signal Pathway; Skin; Sphingolipids; Staging; Surface; System; Testing; Toxic effect; Transferase; Transferase Gene; Vaccines; Vertebral column; Virulence; Visceral; Work; chemotherapy; feeding; fly; gene discovery; genome sequencing; glycosylation; glycosyltransferase; improved; in vivo; inorganic phosphate; inositolphosphorylceramide; insight; lipid metabolism; lipophosphonoglycan; macrophage; man; mutant; novel; positional cloning; programs; success; sugar nucleotide; vaccination strategy; vector

DESCRIPTION (provided by applicant): Leishmaniasis is a major health problem to humans and is caused by the protozoan parasite Leishmania. Depending on the species, Leishmania-induced pathology ranges from self-healing, cutaneous lesions to fatal, visceral diseases. Leishmania express a family of structurally interrelated glycoconjugates, dominated by its surface lipophosphoglycan (LPG), that have critical roles in parasite survival and virulence. The sharing of structural domains among virtually all parasite surface glycoconjugates, however, often leads to imprecision in our understanding of their unique and/or overlapping roles in vivo. Through identification of specific mutants through forward and reverse genetics, and generation of genetically complemented counterparts, we have established a powerful approach enabling us to systematically dissect the biosynthetic pathway of LPG and related glycoconjugates. We plan to focus on mutants defective in a well chosen set of biochemical steps and genes that will allow us to dissect the role of individual glycoconjugates and/or their specific domains in various stages of the infections cycle. The ability of infectious metacyclic parasites to enter, survive and differentiate into amastigotes following ingestion by macrophages will be studied; where possible amastigote initiated infections will be performed as well, as glycoconjugate function can differ greatly in the two infectious stages. The ability of metacyclic promastigotes or amastigotes to induce pathology in susceptible and resistant mice will be studied, and long term persistence evaluated. Lastly, in collaboration with David Sacks we will evaluate the ability of promastigotes fed within a blood meal to survive and develop within the sand fly vector. Several pathways identified show potential for chemotherapy and/or vaccination strategies in the future. Our ultimate goal is a comprehensive understanding of the genes and gene products responsible for synthesizing Leishmania surface and secreted molecules, and their individual and specific roles in parasite virulence. The four specific aims of this competing renewal application are: 1. To identify candidate glycoconjugate(s) that are key virulence molecules responsible for amastigote stage virulence in L. major, as defined by studies of the avirulent, persistent lpg2- mutant. 2. To characterize new families of mannosyl-phosphate transferases and galactosyltransferases involved in synthesis of the phosphoglycan (PG) repeating unit Gal(21,4)Man(11)-PO4- backbone. 3. To determine the role of the emerging sphingolipid (SL) pathway and inositolphosphorylceramide (IPC) in amastigotes. 4. To develop comprehensive Leishmania glycomics. As part of these studies we plan to test whether the recently discovered active RNAi pathway of L. braziliensis may be productively incorporated into the study of Leishmania glycoconjugates.Project Narrative Leishmania are important tropical parasites, causing disease in more than 10 million people worldwide; more than 400 million people are at risk for infection in endemic regions. US military personnel have significant risk of infection in these areas as well. Depending on the species, Leishmania-induced pathology ranges from self-healing, cutaneous lesions to fatal, visceral diseases. Currently, there are no vaccines available against leishmaniasis, and the only approved chemotherapies are marginally effective, difficult to administer, and have significant associated toxicities. The underlying tenet of our research program is that improved understanding of key pathways required for parasite virulence and viability may provide opportunities for the development of improved therapies. Leishmania express a family of structurally interrelated glycoconjugates, dominated by its surface lipophosphoglycan (LPG), that have critical roles in parasite survival and virulence. The sharing of structural domains among virtually all parasite surface glycoconjugates, however, complicates our understanding of their unique and/or overlapping roles in vivo. To overcome this, we use genetic approaches to make parasite mutants altered in specific molecules, or domains, or smaller substitutions. As there are many possible steps, and some of their effects may be similar, we try to choose ones that will give us the greatest information. Then, we test each mutant in the parasite infectious cycle. Leishmania are normally transmitted by the bite of an infected sand fly, so the first step in infection is the deposition of infective metacyclic form parasites into the skin where they are taken up by macrophages. There they resist host defenses and differentiate into another form called amastigotes, which are adapted for replication and go on to cause disease. Eventually sand flies bite infected animals, and the parasite has to survive within the alimentary tract of the fly. We have good assays for testing the effect of each mutant in each of the steps throughout the infectious cycle. We have good success in previous work, and several pathways and molecules already identified show potential for chemotherapy and/or vaccination strategies in the future. Our ultimate goal is a comprehensive understanding of the genes and gene products responsible for synthesizing Leishmania surface and secreted molecules, and their individual and specific roles in parasite virulence.

描述（由申请人提供）：利什曼病是人类的一个主要健康问题，由原生动物寄生虫利什曼原虫引起。根据物种的不同，利什曼原虫引起的病理范围从自愈性皮肤损伤到致命性内脏疾病。利什曼原虫表达一系列结构上相互关联的糖复合物，以其表面脂磷酸聚糖 (LPG) 为主，在寄生虫的生存和毒力中发挥着关键作用。然而，几乎所有寄生虫表面糖缀合物之间共享结构域常常导致我们对其独特和/或重叠的体内作用的理解不精确。通过正向和反向遗传学鉴定特定突变体，以及生成遗传互补的对应物，我们建立了一种强大的方法，使我们能够系统地剖析 LPG 和相关糖复合物的生物合成途径。我们计划重点关注一组精心选择的生化步骤和基因中存在缺陷的突变体，这将使我们能够剖析单个糖复合物和/或其特定结构域在感染周期各个阶段的作用。将研究传染性后循环寄生虫在被巨噬细胞摄入后进入、存活并分化为无鞭毛体的能力；在可能的情况下，也会进行无鞭毛体引发的感染，因为糖缀合物功能在两个感染阶段可能有很大差异。将研究后循环前鞭毛体或无鞭毛体在易感和耐药小鼠中诱导病理的能力，并评估长期持久性。最后，我们将与 David Sacks 合作，评估以血粉喂养的前鞭毛体在白蛉媒介中生存和发育的能力。已确定的几种途径显示出未来化疗和/或疫苗接种策略的潜力。我们的最终目标是全面了解负责合成利什曼原虫表面和分泌分子的基因和基因产物，以及它们在寄生虫毒力中的个体和特定作用。这一竞争性更新申请的四个具体目标是： 1. 鉴定候选糖缀合物，它们是导致大利斯特氏菌无鞭毛体阶段毒力的关键毒力分子，如无毒力、持久性 lpg2-突变体的研究所定义。 2. 表征参与磷酸聚糖 (PG) 重复单元 Gal(21,4)Man(11)-PO4- 主链合成的新甘露糖基磷酸转移酶和半乳糖基转移酶家族。 3. 确定新兴的鞘脂（SL）途径和肌醇磷酸神经酰胺（IPC）在无鞭毛体中的作用。 4. 开发综合利什曼原虫糖组学。作为这些研究的一部分，我们计划测试最近发现的巴西利什曼原虫活性 RNAi 途径是否可以有效地纳入利什曼原虫糖缀合物的研究中。 项目叙述 利什曼原虫是重要的热带寄生虫，导致全世界超过 1000 万人患病；流行地区有超过4亿人面临感染风险。美国军事人员在这些地区也存在很大的感染风险。根据物种的不同，利什曼原虫引起的病理范围从自愈性皮肤损伤到致命性内脏疾病。目前，还没有针对利什曼病的疫苗，唯一批准的化疗方法效果有限，难以施用，并且具有显着的相关毒性。我们研究计划的基本宗旨是，提高对寄生虫毒力和活力所需的关键途径的了解可能为开发改进的疗法提供机会。利什曼原虫表达一系列结构上相互关联的糖复合物，以其表面脂磷酸聚糖 (LPG) 为主，在寄生虫的生存和毒力中发挥着关键作用。然而，几乎所有寄生虫表面糖缀合物之间共享结构域，使我们对其独特和/或重叠的体内作用的理解变得复杂。为了克服这个问题，我们使用遗传方法使寄生虫突变体在特定分子、结构域或较小的替换中发生改变。由于可能的步骤有很多，并且其中一些效果可能相似，因此我们尝试选择能够为我们提供最多信息的步骤。然后，我们测试寄生虫感染周期中的每个突变体。利什曼原虫通常通过受感染的沙蝇叮咬传播，因此感染的第一步是感染性后循环形式寄生虫沉积到皮肤中，并被巨噬细胞吸收。在那里，它们抵抗宿主防御并分化成另一种称为无鞭毛体的形式，这种形式适合复制并继续引起疾病。最终，白蛉会叮咬受感染的动物，而寄生虫必须在白蛉的消化道内生存。我们有很好的分析方法来测试每个突变体在整个感染周期每个步骤中的效果。我们在之前的工作中取得了良好的成功，并且已经确定的几种途径和分子显示出未来化疗和/或疫苗接种策略的潜力。我们的最终目标是全面了解负责合成利什曼原虫表面和分泌分子的基因和基因产物，以及它们在寄生虫毒力中的个体和特定作用。