Identification of a Parasite-Specific, Lipid-Metabolic Pathway Essential for Leis

利什曼原虫在人类身上引起一系列破坏性疾病，称为利什曼病。 目前的药物治疗不充分，往往是有毒的，而且没有安全的疫苗可用。一个主要障碍是 新疗法的发展是对利什曼原虫如何逃避的缺乏了解 免疫激活并持续存在于哺乳动物宿主中。我们的长期目标是阐明 利什曼原虫毒力中的寄生虫脂代谢。与膜蛋白和 在原生动物病原体中，对脂类的生物学研究极少。这个 本应用的目的是确定肌醇周转的分子机制。 磷酰神经酰胺(IPC)与利什曼原虫的感染性有关。IPC是一种特定于寄生虫的 脂类和初步研究表明它的降解(由假定的肌醇磷酸神经酰胺特异性催化 磷脂酶C或IPC-PLC)是利什曼原虫毒力所必需的。中心假设是 IPC-PLC在颠覆宿主免疫防御和生存中发挥着不可或缺的作用 哺乳动物中的无鞭毛体。这个项目的基本原理是，这种新知识可能会导致 用于选择性化疗治疗利什曼原虫的寄生虫特异性靶点的确定。以Strong为指导 初步数据，将通过追求四个具体目标来检验中心假设：1)评估 IPC-PLC对前鞭毛体毒力性状表达的影响2)分子检测 IPC-PLC零前突起与哺乳动物宿主的相互作用；3)评估 IPC-PLC在利什曼原虫毒力中的阶段和细胞器特异性表达；4)纯化IPC-PLC和 测定其生化性质。在第一和第二个目标下，IPC-PLC的一个零突变体(已经 )将被用来揭示IPC退化对建立和持续的贡献 巨噬细胞和小鼠的利什曼原虫感染。在第三个目标下，IPC-PLC的活动将是 在严格的空间或时间控制下引入零突变体；这种阶段的影响-和 IPC-PLC在毒力上的细胞器特异性表达将在前鞭毛体和无鞭毛体中进行评估。 根据最终目标，IPC-PLC将被提纯为重组蛋白(从大肠杆菌中)或作为 将测定内源蛋白(来自利什曼原虫)及其生化特性。这个 拟议的研究具有创新性和非常重要的意义，因为它有望首次确定 时间，丰富的膜脂(IPC)的降解和毒力之间的关键联系 利什曼原虫。此外，由于IPC和IPC-PLC在人类中不存在，这样的发现 预计将确定和验证选择性治疗的新靶点，以控制这些危险 病原体。

Leishmania parasites cause a spectrum of devastating diseases in humans known as leishmaniasis. Current drug treatments are inadequate, often toxic, and no safe vaccine is available. A major obstacle in the development of new therapies is the lack of understanding on how Leishmania parasites evade immune activation and persist in mammalian hosts. The long-term goal is to elucidate the roles of parasite lipid metabolism in Leishmania virulence. In comparison to membrane proteins and glycoconjugates, the biology of lipids has been extremely understudied in protozoan pathogens. The objective of this application is to determine the molecular mechanism by which the turnover of inositol phosphorylceramide (IPC) contributes to the infectivity of Leishmania parasites. IPC is a parasite-specific lipid and preliminary studies suggest its degradation (catalyzed by a putative inositol phosphorylceramidespecific phospholipase C or IPC-PLC) is essential for Leishmania virulence. The central hypothesis is that IPC-PLC plays indispensable roles in the subversion of host immune defense and the survival of amastigotes in mammals. The rationale for this project is that such new knowledge may lead to the identification of parasite-specific targets for selective chemotherapy against Leishmania. Guided by strong preliminary data, the central hypothesis will be tested by pursuing four specific aims: 1) evaluate the impact of IPC-PLC on the expression of virulent traits in promastigotes; 2) determine the molecular interaction between IPC-PLC null promastigotes and mammalian hosts; 3) evaluate the importance of stage- and organelle-specific expression of IPC-PLC in Leishmania virulence; and 4) purify IPC-PLC and determine its biochemical properties. Under the first and second aims, a null mutant of IPC-PLC (already in hand) will be used to unmask the contribution of IPC degradation to the establishment and persistence of Leishmania infection in macrophages and mice. Under the third aim, the activity of IPC-PLC will be introduced into the null mutant under strict spatial or temporal control; effects of such stage- and organelle-specific expression of IPC-PLC on virulence will be evaluated in promastigotes and amastigotes. Under the final aim, IPC-PLC will be purified either as a recombinant protein (from E. coli) or as an endogenous protein (from Leishmania parasites) and its biochemical properties will be determined. The proposed research is innovative and highly significant, because it is expected to identify, for the first time, a crucial link between the degradation of an abundant membrane lipid (IPC) and virulence in Leishmania parasites. In addition, because IPC and IPC-PLC are not present in humans, such findings are expected to identify and validate new targets for selective therapy to control these dangerous pathogens.