Genetic dissection of parasite metabolism

描述（由申请人提供）：本提案概述了从基因上剖析人类病原体弓形虫脂肪酸代谢的综合计划。T.弓形虫感染在美国很普遍（22%的人口是慢性感染），并且虽然通常是良性的，但在免疫抑制的个体（例如，患有HIV-AIDS的个体、移植受体或血液恶性肿瘤的个体）中可引起危及生命的疾病。先天性T.弓形虫也是一个主要的公共卫生问题。最近已确定高度致命的寄生虫菌株是严重和复发性眼部感染的原因，最终导致失明。T.弓形虫也有可能引起重大的水传播疾病暴发，并已被CDC列为潜在的生物恐怖主义病原体（附录B）。目前可用的治疗具有频繁和显著的副作用，并且在慢性感染中没有显示出有效性，从而允许活动性感染复发。因此，迫切需要新的药物。在顶复门寄生虫中发现的叶绿体样细胞器为药物开发提供了几个有希望的寄生虫特异性靶向途径。在这些途径中，有一种细菌II型脂肪酸合成途径，该途径中的酶一直是药物化学努力的主题，以开发抗疟疾和弓形虫病的药物。然而，该通路对T.弓形虫和相关的顶复门寄生虫仍不清楚。此外，寄生虫基因组编码额外的酶系统，这些酶系统可能通过合成或从宿主细胞中回收来提供脂肪酸。需要详细了解这些途径的功能和相对重要性，以指导药物开发工作最有前途的目标。在这个项目中，我们将使用遗传学和代谢组学来剖析三个单独的途径复杂的相互作用。使用一种新颖高效的方法来设计条件T。弓形虫突变体我们将在体内严格测试每个单独途径的重要性和功能。我们将使用无偏的代谢组学分析来确定特定途径的丧失对寄生虫脂肪酸和脂质组成的影响。为了确定各个途径之间的相互作用和寄生虫与其宿主细胞之间的相互作用，我们使用稳定的表位追踪进行代谢通量研究。总的来说，我们希望概述的研究产生一个详细的机制了解脂肪酸合成作为寄生虫代谢和代谢宿主-寄生虫相互作用的重要组成部分。突变体分析将突出真正重要的成分作为潜在的药理学靶点。我们也期望，沿着磨练的遗传学和代谢组学工具，将被证明对解剖寄生虫生物学的许多方面非常有用，而不仅仅是脂质代谢。

DESCRIPTION (provided by applicant): This proposal outlines a comprehensive plan to genetically dissect the fatty acid metabolism of the human pathogen Toxoplasma gondii. T. gondii infection is widespread in the U.S. (22% of the population is chronically infected) and while usually benign can cause life-threatening disease in immunosuppressed individuals (e.g. those with HIV-AIDS, transplant recipients, or hematological malignancies). Congenital transmission of T. gondii is also a major public health concern. Highly virulent parasite strains have been recently identified as the cause of severe and recurring eye infections that ultimately lead to blindness. T. gondii also has the potential to cause significant waterborne outbreaks and has been listed by the CDC as a potential bioterrorism pathogen (appendix B). The currently available treatment has frequent and significant adverse effects and shows no efficacy in chronic infection, thus allowing for recrudescence of the active infection. Thus, new drugs are urgently needed. The discovery of a chloroplast-like organelle in apicomplexan parasites provides several promising parasite-specific target pathways for drug development. Among these pathways is a bacterial type II fatty acid synthesis pathway, and enzymes in this pathway have been the subject of intensive medicinal chemistry efforts to develop drugs against malaria and toxoplasmosis. However, what the precise function of this pathway for T. gondii and related apicomplexan parasites is remains unclear. Furthermore, the parasite genome encodes additional enzyme systems that might supply fatty acids either by synthesis or salvage from the host cell. A detailed understanding of the function and relative importance of these pathways is needed to guide the drug development effort to the most promising targets. In this project we will use genetics and metabolomics to dissect the complex interaction of three individual pathways. Using a novel and highly efficient approach to engineer conditional T. gondii mutants we will rigorously test the importance and function of each individual pathway in vivo. We will determine the impact of the loss of specific pathways on the parasite fatty acid and lipid composition using unbiased metabolomic profiling. To define the interactions between individual pathways and between the parasite and its host cell we conduct metabolic flux studies using stable epitope tracing. Overall we expect the outlined studies to produce a detailed mechanistic understanding of fatty acid synthesis as an important part of parasite metabolism and metabolic host-parasite interaction. Mutant analysis will highlight truly essential components as potential pharmacological targets. We also expect that the genetic and metabolomic tools honed along the way will prove highly useful for the dissection of many facets of parasite biology beyond lipid metabolism.