CHARACTERIZATION OF ESSENTIAL RHOPTRY KINASES OF TOXOPLASMA GONDII

描述（由申请人提供）：刚地弓形虫是一种广泛存在的原生动物寄生虫，能够引起发育中的婴儿先天性疾病和免疫功能低下患者的严重并发症。近三分之一的人口被弓形虫慢性感染。目前的治疗方法无法治愈慢性感染，由于毒性，长期治疗往往导致不耐受。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制定合理的药物设计策略。弓形虫在广泛的温血宿主中非常有效地寄生，并且可以感染几乎任何有核细胞类型。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（称为异质体）注入宿主细胞质。结果是，宿主用来对抗传染性寄生虫的许多众所周知的先天机制变得无反应或不起作用。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利用，使弓形虫能够畅通无阻地完成其细胞内生命周期。核糖核酸细胞器的蛋白质组学分析确定了一个高度扩展的丝氨酸/苏氨酸（S/T）激酶家族（ROP激酶）的存在，毒力基因的遗传作图研究表明其中一些是关键的毒力因子。在弓形虫基因组中发现了大约20种活性ROP激酶，系统发育、结构和功能分析表明，它们与人类中发现的任何主要S/T激酶家族都有显著差异。几个多态ROP激酶已经被证明可以直接调节寄生虫的毒力，但令人惊讶的是，没有一个是寄生虫生存所必需的。该应用程序的目的是通过系统地确定它们在寄生虫生存能力和毒力中的作用，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，并表征蛋白敲低对体外细胞内生命周期的表型影响以及小鼠模型中的毒力。必需的ROP激酶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激酶复合物的分离和宿主底物的捕获将通过深入了解可能在调节激酶功能或决定底物特异性中起作用的其他因素来完成分析。总之，该项目将整合各种表型分析，宿主细胞转录谱和ROP激酶复合物的蛋白质组学特征，以产生必需ROP激酶在关键宿主-病原体相互作用中发挥作用的综合观点。更重要的是，由此产生的分析可能会确定许多新的潜在治疗靶点。

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.