CHARACTERIZATION OF ESSENTIAL RHOPTRY KINASES OF TOXOPLASMA GONDII

描述（由申请方提供）：弓形虫是一种分布广泛的原生动物寄生虫，能够引起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。近三分之一的人口是慢性感染T。刚地。目前的疗法无法治愈慢性感染和不耐受，由于毒性，往往是长期治疗的结果。我们有效治疗T.弓形虫感染需要鉴定新的治疗靶点和开发合理的药物设计策略。T.弓形虫在广泛的温血宿主中寄生非常有效，并且可以感染几乎任何有核细胞类型。在入侵过程中T.弓形虫将来自分泌细胞器（称为棒状体）的蛋白质的异质混合物注入宿主细胞质中。其结果是，许多众所周知的先天机制所采用的主机，以打击传染性寄生虫成为无反应或瘫痪。宿主细胞凋亡、代谢物隔离和免疫反应的途径变得有效地被选择，使得T。弓形虫完成其细胞内的生命周期畅通无阻。棒状体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（ROP激酶）家族的存在和毒力基因的遗传作图研究牵连其中几个关键毒力因子。在T.弓形虫基因组和系统发育、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。几种多态性ROP激酶已被证明直接调节寄生虫毒力，但令人惊讶的是，没有一种是寄生虫生存所必需的。本申请的目的是通过系统地确定ROP激酶家族在寄生虫生存力和毒力中的作用，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，并表征蛋白质敲低对体外细胞内生命周期的表型效应以及小鼠模型中的毒力。将通过基于微阵列的感染后发生的转录变化的比较来评估必需ROP激酶在宿主细胞调节中的作用。天然核心ROP激酶复合物的分离和宿主底物的捕获将通过提供对可能在调节激酶功能或确定底物特异性中起作用的额外因素的洞察来完成分析。总之，该项目将整合各种表型分析，宿主细胞转录谱，ROP激酶复合物的蛋白质组学表征，以产生一个复合的观点，重要的ROP激酶在关键的宿主-病原体相互作用中发挥的作用。更重要的是，由此产生的分析可能会识别出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.