CHARACTERIZATION OF ESSENTIAL RHOPTRY KINASES OF TOXOPLASMA GONDII

描述(申请人提供)：弓形虫是一种分布广泛的原虫寄生虫，能够导致发育中婴儿的先天性疾病和免疫功能低下患者的严重并发症。近三分之一的人口长期感染弓形虫。目前的治疗方法无法治愈慢性感染和不耐受，因为毒性往往是长期治疗的结果。我们有效治疗弓形虫感染的能力需要确定新的治疗靶点和开发合理的药物设计策略。弓形虫在寄生广泛的温血宿主方面非常有效，几乎可以感染任何有核细胞类型。在入侵过程中，弓形虫将一种来自分泌细胞器的异质蛋白质混合物注入宿主细胞质，这种混合物被称为棒状体。其结果是，宿主用来对抗传染性寄生虫的许多众所周知的固有机制变得反应迟钝或不起作用。参与细胞凋亡、代谢物隔离和免疫反应的宿主途径被有效地增选，从而使弓形虫不受阻碍地完成其细胞内生命周期。对杆状细胞器的蛋白质组学分析证实，存在一个高度扩展的丝氨酸/苏氨酸(S/T)激酶家族(ROP激酶)，毒力基因的遗传图谱研究表明，其中几个是关键的毒力因子。目前已在弓形虫基因组中鉴定出约20个活性的ROP激酶，系统发育、结构和功能分析表明，它们与在人类中发现的任何一个主要的S/T激酶家族都有显著的不同。自那以后，几种多态的ROP激酶被证明直接调节寄生虫的毒力，但令人惊讶的是，没有一种对寄生虫的生存是必不可少的。这项应用的目的是通过系统地确定它们在寄生虫生存和毒力中的作用，提供对ROP激酶家族的全面分析。我们将首先定义激酶家族的基本成员，并在体外表征蛋白质敲除对细胞内生命周期的表型效应以及在小鼠模型中的毒力。必要的ROP蛋白在宿主细胞调节中的作用将通过基于微阵列的感染后转录变化的比较来评估。天然核心ROP激酶复合体的分离和宿主底物的捕获将提供对可能在调节激酶功能或确定底物特异性方面发挥作用的额外因素的洞察，从而完成分析。总而言之，该项目将整合各种表型分析、宿主细胞转录图谱和ROP激酶复合体的蛋白质组学特征，以产生基本ROP激酶在关键宿主-病原体相互作用中所起作用的综合视图。更重要的是，由此产生的分析可能会确定一系列新的潜在治疗靶点。

DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread protozoan parasite that is capable of causing congenital disease in developing infants and severe complications in immunocompromised patients. Nearly a third of the human population is chronically infected by T. gondii. Current therapies are unable to cure chronic infection and intolerance, due to toxicity, often results from long-term treatment. Our ability to effectively treat T. gondii infection requires the identification of new therapeutic targets and the development of a rational drug design strategy. T. gondii is highly effective at parasitizing a broad range of warm-blooded hosts and can infect nearly any nucleated cell type. During the invasion process T. gondii injects a heterogeneous mixture of proteins from secretory organelles, known as rhoptries, into the host cytoplasm. The result is that many of the well known innate mechanisms employed by hosts to combat infectious parasites become unresponsive or inoperative. Host pathways involved in apoptosis, metabolite sequestering, and the immune response become effectively co-opted allowing T. gondii to complete its intracellular life cycle unimpeded. Proteomic analysis of the rhoptry organelles identified the presence of a highly expanded family of serine/threonine (S/T) kinases (ROP kinases) and genetic mapping studies of virulence genes implicated several of these as critical virulence factors. Approximately 20 active ROP kinases have been identified in the T. gondii genome and phylogenetic, structural, and functional analyses have indicated that they differ significantly from any of the major families of S/T kinases found in humans. Several polymorphic ROP kinases have since been shown to directly modulate parasite virulence yet, surprisingly, none were essential for parasite viability. The goal of this application is to provide a comprehensive analysis of the ROP kinase family by systematically determining their role in parasite viability and virulence. We will first define the essential members of the kinase family and characterize the phenotypic effects of protein knockdown on the intracellular life cycle in vitro as well as virulence in the mouse model. The role of the essential ROP kinases in host cell modulation will be assessed through microarray based comparison of transcriptional changes occurring after infection. The isolation of native core ROP kinase complexes and the trapping of host substrates will complete the analysis by providing insight into additional factors which may play a role in regulating kinase function or determining substrate specificity. In summary this project will integrate various phenotypic analyses, host cell transcriptional profiling, and proteomic characterization of ROP kinase complexes to yield a composite view of the role that essential ROP kinases play in critical host-pathogen interactions. More importantly the resulting analyses may identify a host of new potential therapeutic targets.