Genetic dissection of parasite metabolism

这项建议概述了一个全面的计划，从基因上剖析人类的脂肪酸代谢， 病原体弓形虫T.弓形虫感染在美国很普遍（22%的人口是慢性的， 感染），而通常是良性的，可导致免疫抑制个体的危及生命的疾病（例如， 患有HIV-AIDS、移植受者或恶性血液病的患者）。先天性T. 弓形虫病也是一个重大的公共卫生问题。高毒力寄生虫株最近被鉴定为 导致严重和复发性眼部感染，最终导致失明。T.弓形虫也有可能 导致重大的水传播疾病爆发，并已被CDC列为潜在的生物恐怖主义病原体 （附录B）。目前可用的治疗方法具有频繁和显著的不良反应， 在慢性感染中有效，从而允许活动性感染复发。因此，新药物 迫切需要。在顶复门寄生虫中发现的叶绿体样细胞器提供了几个 有希望的寄生虫特异性靶向药物开发途径。在这些途径中，有一种细菌II型 脂肪酸合成途径和该途径中的酶已经成为深入医学研究的主题。 在化学方面的努力，以开发抗疟疾和弓形虫病的药物。然而， 这条通路对于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脂肪酸中回收来提供脂肪酸。 宿主细胞需要详细了解这些途径的功能和相对重要性， 将药物开发工作引向最有希望的目标。在这个项目中，我们将使用遗传学和 代谢组学来剖析三个单独途径的复杂相互作用。采用新颖高效的 工程条件T.我们将严格测试每个弓形虫突变体的重要性和功能 体内的单个通路。我们将确定特定途径的损失对寄生虫脂肪的影响。 酸和脂质组成，使用公正的代谢组学分析。来定义个体之间的相互作用 途径和寄生虫及其宿主细胞之间，我们使用稳定表位进行代谢通量研究 追踪总的来说，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。突变分析 将突出真正重要的成分作为潜在的药理学靶点。我们还认为， 代谢组学工具沿着磨练，将被证明是非常有用的解剖的许多方面， 寄生虫生物学超越脂质代谢。

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.