Glycosylation Mutants of Leishmania

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，利什曼病诱导的病理学范围从自愈性皮肤病变到致命的内脏疾病。利什曼原虫表达一个家族的结构相关的糖缀合物，占主导地位的表面脂磷酸聚糖（LPG），在寄生虫的生存和毒力的关键作用。然而，几乎所有寄生虫表面糖缀合物之间的结构域的共享，往往导致我们在体内的独特和/或重叠的作用的理解不精确。通过正向和反向遗传学鉴定特定突变体，并产生遗传互补的对应物，我们已经建立了一个强大的方法，使我们能够系统地剖析LPG和相关糖缀合物的生物合成途径。我们计划专注于突变体缺陷的一组精心挑选的生化步骤和基因，这将使我们能够解剖的作用，个别糖缀合物和/或其特定的结构域在感染周期的各个阶段。将研究巨噬细胞摄入后感染性亚环寄生虫进入、存活和分化为无鞭毛体的能力;如果可能，也将进行无鞭毛体启动的感染，因为糖缀合物功能在两个感染阶段可能有很大差异。将研究代谢环前鞭毛体或无鞭毛体在易感和抗性小鼠中诱导病理学的能力，并评价长期持久性。最后，我们将与大卫萨克斯合作，评估前鞭毛体在血餐中生存和发育的能力。确定的几种途径显示了未来化疗和/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。这一竞争性续期申请的四个具体目标是：1。目的鉴定与无鞭毛体阶段致病力相关的关键毒力分子候选糖缀合物。主要的，如无毒力的持久性LPG 2突变体的研究所定义的。2.表征参与磷酸聚糖（PG）重复单元Gal（21，4）Man（11）-PO 4-骨架合成的甘露糖基磷酸转移酶和半乳糖基转移酶的新家族。3.确定新出现的鞘脂（SL）途径和肌醇磷酸神经酰胺（IPC）在无鞭毛体中的作用。4.建立全面的利什曼原虫糖组学。作为这些研究的一部分，我们计划测试最近发现的L。巴西利什曼原虫可能被有效地纳入利什曼原虫糖缀合物的研究。 利什曼原虫是重要的热带寄生虫，在全球范围内导致超过1000万人患病;在流行地区，超过4亿人面临感染风险。美国军事人员在这些地区也有很大的感染风险。根据物种的不同，利什曼病诱导的病理学范围从自愈性皮肤病变到致命的内脏疾病。目前，还没有针对利什曼病的疫苗，唯一批准的化疗药物效果不佳，难以管理，并具有显着的相关毒性。我们的研究计划的基本宗旨是，提高对寄生虫毒力和生存能力所需的关键途径的理解，可能会为开发改进的疗法提供机会。利什曼原虫表达一个家族的结构相关的糖缀合物，占主导地位的表面脂磷酸聚糖（LPG），在寄生虫的生存和毒力的关键作用。然而，几乎所有寄生虫表面糖缀合物之间的结构域的共享，使我们对它们在体内的独特和/或重叠作用的理解变得复杂。为了克服这一点，我们使用遗传方法使寄生虫突变体在特定分子或结构域或更小的取代中发生改变。由于有许多可能的步骤，其中一些效果可能是相似的，我们试图选择那些能给我们最大信息的步骤。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被感染的白蛉的叮咬传播，因此感染的第一步是感染性亚循环形式寄生虫沉积到皮肤中，在那里它们被巨噬细胞吸收。在那里，它们抵抗宿主的防御，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。最终沙蝇叮咬受感染的动物，寄生虫必须在苍蝇的消化道内生存。我们有很好的方法来测试每个突变体在整个感染周期的每个步骤中的作用。我们在以前的工作中取得了很好的成功，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。

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.