GLOBAL ANALYSIS OF THE HOST CELL RESPONSE TO TOXOPLASMA GONDII INFECTION IN ASTR

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 弓形虫是中枢神经系统的一种主要机会性病原体，在艾滋病患者和其他免疫功能受损的人中会导致重大疾病，如移植受者和接受化疗的癌症患者。在这些人中，感染是由于大脑中的潜伏感染重新激活，并导致严重的、往往是致命的坏死性脑炎。细胞因子在弓形虫中枢神经系统的调控中起着重要作用，而干扰素-γ(IFNv)是控制弓形虫在脑和其他组织中复制的主要细胞因子。IFNy对小鼠星形胶质细胞和巨噬细胞的抑制机制部分依赖于IFNv诱导的反应蛋白IGTP。自然的宿主防御机制受到寄生虫的影响，众所周知，寄生虫可以显著改变宿主细胞的转录，而且有明确的证据表明，寄生虫积极干预了影响宿主细胞凋亡和细胞因子反应的途径。这场拉锯战的生化结果影响了宿主细胞环境的建立，这种环境支持或敌视寄生虫的生长和发育。宿主细胞的组成和寄生虫操纵宿主细胞环境的因素尚不清楚，但这些机制似乎在表达广泛生长和毒力表型的菌株之间存在很大差异。星形胶质细胞是弓形虫在脑内的重要宿主细胞，也是重要的干扰素激活的效应细胞，在脑内介导对弓形虫的抵抗。因此，星形胶质细胞模型提供了一个机会来了解宿主防御和寄生虫在临床相关细胞类型中的生存机制。我们将采用一种综合的方法来表征在本项目中IFNy刺激提供保护的条件下，从体外暴露于弓形虫感染的动物获得的原代星形胶质细胞中宿主mRNA和蛋白质表达的变化以及蛋白质翻译后修饰的变化。我们将定义星形胶质细胞中的IFNy反应，并探索具有不同遗传谱系和毒力表型的寄生虫如何改变这些宿主细胞的变化。这项建议的三个具体目的是：1)在IFNy刺激和未刺激的星形胶质细胞中转录分析宿主细胞对弓形虫感染的反应，2)在IFNy刺激和未刺激的星形胶质细胞中对宿主细胞对弓形虫感染的反应进行蛋白质组学分析，以及3)建立弓形虫在星形胶质细胞中受IFNy刺激的存活突变体的筛选。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Toxoplasma gondii is a major opportunistic pathogen of the central nervous system, that causes significant disease in AIDS patients and other immunocompromised individuals, such as transplant recipients and cancer patients undergoing chemotherapy. In these individuals infection is due to reactivation of a latent infection in the brain and results in severe and often fatal necrotizing encephalitis. Cytokines play an important role in the regulation of T. gondii in the central nervous system and interferon-gamma (IFNv) is the main cytokine controlling replication of T. gondii in the brain and in other tissues. The mechanism of IFNy inhibition in murine astrocytes and macrophages is partially dependent upon the IFNv-induced response protein, IGTP. Natural host-defense mechanisms are influenced by the parasite, which is known to substantially alter host cell transcription, and there is clear evidence for active parasite intervention in pathways affecting host cell apoptosis and the cytokine response. The biochemical outcome of this tug-of-war influences the establishment of a host cell environment that either supports or is hostile to parasite growth and development. The host cell components and the factors by which the parasite manipulates the host cell environment are not understood, but these mechanisms appear to vary extensively between strains that express a wide range of growth and virulent phenotypes. Astrocytes are an important host cell for T. gondii in the brain and an important IFNy-activated effector cell, mediating resistance to T. gondii in the brain. As such, the astrocyte model provides an opportunity to understand host-defense and parasite survival mechanisms in a clinically relevant cell type. We will take a comprehensive approach to characterize the changes in host mRNA and protein expression and protein post-translational modification that occur in primary astrocytes obtained from animals that are exposed in vitro to Toxoplasma infection, under conditions where protection is afforded by IFNy stimulation in this project. We will define the IFNy response in astrocytes and explore how these host cell changes are altered by parasites of distinct genetic lineage and virulent phenotypes. The three specific aims of this proposal are: 1) Transcriptional analysis of the host cell response to T. gondii infection in IFNy stimulated vs. unstimulated astrocytes, 2) Proteomic analysis of the host cell response to T. gondii infection in IFNy stimulated vs. unstimulated astrocytes and 3) Development of a screen for T. gondii survival mutants subjected to IFNy stimulation in astrocytes.