Glycosylation Mutants of Leishmania

描述（由申请人提供）：利什曼病是人类的主要健康问题，由原虫寄生虫利什曼原虫引起。根据物种的不同，利什曼引起的病理从自愈、皮肤病变到致命的内脏疾病不等。利什曼原虫表达一个结构相关的糖缀合物家族，以其表面脂磷聚糖（LPG）为主，在寄生虫的生存和毒力中起关键作用。然而，几乎所有寄生虫表面糖缀合物的结构域共享往往导致我们对它们在体内独特和/或重叠作用的理解不精确。通过正向和反向遗传学鉴定特定突变体，并生成遗传互补对应体，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和相关糖缀合物的生物合成途径。我们计划将重点放在一组精心选择的生化步骤和基因中的突变缺陷上，这将使我们能够解剖单个糖缀合物和/或其特定结构域在感染周期的各个阶段的作用。将研究传染性元环寄生虫在被巨噬细胞吞噬后进入、存活并分化为无尾线虫的能力；在可能的情况下，由于糖结合功能在两个感染阶段可能有很大的不同，无尾线虫也会引起感染。在易感小鼠和耐药小鼠中，将研究微环原毛菌或无尾毛菌诱导病理的能力，并评估其长期持久性。最后，与David Sacks合作，我们将评估在血餐中喂养的promastigotes在沙蝇载体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。这个竞争性更新申请的四个具体目标是：1。通过对无毒的持久性lpg2-突变体的研究，确定在L. major中作为关键毒力分子的候选糖缀合物(s)。2. 表征参与合成磷酸聚糖（PG）重复单元Gal(21,4)Man(11)- po4 -骨架的甘露糖基磷酸转移酶和半乳糖基转移酶的新家族。3. 目的：探讨鞘脂（SL）通路和肌醇磷酸化神经酰胺（IPC）在无尾线虫中的作用。4. 发展全面的利什曼糖组学。作为这些研究的一部分，我们计划测试最近发现的巴西乳杆菌活性RNAi途径是否可以有效地纳入利什曼原虫糖缀合物的研究。项目的叙述

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.