CHARACTERIZATION OF ESSENTIAL RHOPTRY KINASES OF TOXOPLASMA GONDII

弓形虫必需的棒状体激酶的特征

• 批准号: 8490510
• 负责人: RONALD DREW ETHERIDGE
• 金额: $ 5.22万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8490510
• 关键词: Ablation; Acute; Affect; Affinity Chromatography; Apical; Apoptosis; Binding; Biological Assay; Blood; Cell Line; Cell physiology; Cells; Cessation of life; Chemicals; Chromosome Mapping; Chronic; Complex; Core Protein; Cytoplasm; Development; Disease; Drug Design; Family; Family member; Fetus; Fibroblasts; Gene Targeting; Genes; Genome; Goals; Growth; Health; Human; Immune; Immune response; Immune system; Immunocompromised Host; In Vitro; Individual; Infant; Infection; Knock-out; Life; Life Cycle Stages; Mass Spectrum Analysis; Metabolism; Methods; Mus; Nature; One-Step dentin bonding system; Organelles; Parasites; Pathway interactions; Patients; Pharmacotherapy; Phosphotransferases; Phylogenetic Analysis; Play; Population; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Refractory; Relative (related person); Role; Substrate Specificity; System; Testing; Toxic effect; Toxoplasma gondii; Toxoplasmosis; Vacuole; Virulence; Virulence Factors; Work; XRCC5 gene; base; cell type; combat; drug intolerance; extracellular; improved; in vivo; insight; macrophage; member; mouse model; new therapeutic target; novel; parasitism; pathogen; protein complex; rhoptry; secretory protein; therapeutic target

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.

描述（由申请人提供）：弓形虫是一种广泛分布的原生动物寄生虫，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。近三分之一的人口长期感染弓形虫。目前的疗法无法治愈慢性感染，并且由于毒性，通常是长期治疗导致的不耐受。我们有效治疗弓形虫感染的能力需要确定新的治疗靶点并制定合理的药物设计策略。弓形虫在寄生多种温血宿主方面非常有效，并且可以感染几乎任何有核细胞类型。在入侵过程中，弓形虫将来自分泌细胞器（称为棒状体）的异质蛋白质混合物注入宿主细胞质。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变得有效地共同选择，使弓形虫能够不受阻碍地完成其细胞内生命周期。对棒状细胞器的蛋白质组学分析发现存在高度扩展的丝氨酸/苏氨酸 (S/T) 激酶 (ROP 激酶) 家族，并且毒力基因的遗传图谱研究表明其中一些是关键毒力因子。弓形虫基因组中已鉴定出大约 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 激酶家族显着不同。此后，一些多态性 ROP 激酶已被证明可以直接调节寄生虫的毒力，但令人惊讶的是，没有一种激酶对于寄生虫的生存能力至关重要。本应用的目标是通过系统地确定 ROP 激酶家族在寄生虫活力和毒力中的作用，对 ROP 激酶家族进行全面分析。我们将首先定义激酶家族的重要成员，并表征蛋白质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。重要的 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确定底物特异性方面发挥作用的其他因素来完成分析。总之，该项目将整合 ROP 激酶复合物的各种表型分析、宿主细胞转录谱和蛋白质组学表征，以综合了解必需的 ROP 激酶在关键宿主-病原体相互作用中所起的作用。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。