Genetic dissection of parasite metabolism

描述(由申请人提供)：这项建议概述了一个全面的计划，从基因上剖析人类病原体弓形虫的脂肪酸新陈代谢。弓形虫感染在美国很普遍(22%的人口是慢性感染者)，虽然通常是良性的，但也会在免疫抑制的人(如艾滋病毒/艾滋病患者、移植受者或血液系统恶性肿瘤患者)中导致危及生命的疾病。弓形虫的先天性传播也是一个主要的公共卫生问题。最近，高度毒力的寄生虫菌株被确定为严重和反复发生的眼部感染的原因，这些感染最终导致失明。弓形虫也有可能引起重大的水传播暴发，并已被疾控中心列为潜在的生物恐怖主义病原体(附录B)。目前可用的治疗方法具有频繁和显著的不良反应，对慢性感染没有疗效，因此允许活动性感染复发。因此，迫切需要新的药物。顶端复合体寄生虫中叶绿体样细胞器的发现为药物开发提供了几条有希望的寄生虫特异性靶向途径。在这些途径中，有一条是细菌II型脂肪酸合成途径，该途径中的酶一直是密集的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，这条通路对弓形虫和相关的顶端复合体寄生虫的确切功能仍不清楚。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回收提供脂肪酸。需要详细了解这些途径的功能和相对重要性，以指导药物开发努力达到最有希望的目标。在这个项目中，我们将使用遗传学和代谢组学来剖析三个个体通路的复杂相互作用。使用一种新颖而高效的方法来设计条件弓形虫突变体，我们将在体内严格测试每一条单独途径的重要性和功能。我们将使用无偏见的代谢组谱来确定特定途径的丢失对寄生虫脂肪酸和脂肪组成的影响。为了确定单个途径之间以及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。总体而言，我们希望概述的研究将产生对脂肪酸合成作为寄生虫新陈代谢和新陈代谢宿主-寄生虫相互作用的重要部分的详细机制的理解。突变分析将突出真正必要的成分作为潜在的药理靶点。我们还预计，在此过程中磨练的遗传和代谢学工具将被证明对剖析寄生虫生物学的许多方面非常有用，而不仅仅是脂肪代谢。

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.