Glycosylation Mutants of Leishmania

描述(申请人提供)：利什曼病是人类的主要健康问题，由原生动物寄生虫利什曼原虫引起。根据物种的不同，利什曼病引起的病理范围从自我愈合的皮肤损害到致命的内脏疾病。利什曼原虫表达一系列结构上相互关联的糖结合物，主要由其表面的脂磷脂多糖(LPG)主导，在寄生虫的生存和毒力中起着关键作用。然而，几乎所有寄生虫表面糖共轭结构域的共享往往导致我们对它们在体内的独特和/或重叠作用的不精确理解。通过正向和反向遗传学鉴定特定的突变体，以及产生遗传互补的突变体，我们已经建立了一种强大的方法，使我们能够系统地剖析LPG和相关糖结合物的生物合成途径。我们计划专注于在一套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结合物和/或其特定结构域在感染周期的不同阶段的作用。将研究传染性准循环寄生虫进入、存活和被巨噬细胞摄取后分化为无鞭毛体的能力；在可能的情况下，也将进行无鞭毛体启动感染，因为糖结合功能在两个感染阶段可能有很大差异。将研究超环前鞭毛体或无鞭毛体在敏感和耐药小鼠中诱导病理的能力，并评估长期持久性。最后，我们将与大卫·萨克斯合作，评估在血粉中喂养的前鞭毛体在沙蝇媒介中生存和发育的能力。已确定的几条途径显示了未来化疗和/或疫苗接种战略的潜力。我们的最终目标是全面了解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单独和特定作用。这一竞争性更新应用的四个具体目标是：1.通过对无毒、持久的lpg2突变体的研究，确定候选糖共轭化合物(S)是导致大斑潜蝇无鞭毛体阶段毒力的关键毒力分子。2.鉴定参与磷酸多糖(PG)重复单元Gal(21，4)Man(11)-PO4-骨架合成的甘露糖磷酸转移酶和半乳糖基转移酶新家族。3.探讨神经鞘脂(SL)途径和肌醇磷酰神经酰胺(IPC)在无鞭毛体发育中的作用。4.开发综合性利什曼原虫血糖制剂。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共轭化合物的研究。 利什曼原虫是重要的热带寄生虫，导致全球1000多万人患病；在流行地区有4亿多人面临感染风险。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，利什曼病引起的病理范围从自我愈合的皮肤损害到致命的内脏疾病。目前，还没有针对利什曼病的疫苗，唯一获得批准的化疗药物是微效的、难以管理的，并且有显著的相关毒性。我们研究计划的基本原则是，对寄生虫毒力和生存所需关键途径的更好理解可能为改进疗法的发展提供机会。利什曼原虫表达一系列结构上相互关联的糖结合物，主要由其表面的脂磷脂多糖(LPG)主导，在寄生虫的生存和毒力中起着关键作用。然而，几乎所有寄生虫表面糖共轭结构域的共享，使我们对它们在体内的独特和/或重叠作用的理解变得更加复杂。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的分子、结构域或较小的替换中发生改变。由于有许多可能的步骤，而且它们的一些效果可能是相似的，我们试图选择能给我们提供最大信息的步骤。然后，我们测试寄生虫感染周期中的每个突变体。利什曼原虫通常是通过被感染的沙蝇叮咬传播的，因此感染的第一步是将具有感染性的准环状寄生虫沉积到皮肤中，在那里它们被巨噬细胞吸收。在那里，它们抵抗宿主防御，分化成另一种形式，称为无鞭毛体，适合复制并继续致病。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。我们有很好的分析方法来测试每个突变体在整个感染周期的每一步中的影响。我们在以前的工作中取得了良好的成功，已经确定的几种途径和分子显示了未来化疗和/或疫苗策略的潜力。我们的最终目标是全面了解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单独和特定作用。

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.