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: 8202521
• 负责人: RONALD DREW ETHERIDGE
• 金额: $ 4.84万
• 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/8202521
• 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. PUBLIC HEALTH RELEVANCE: Nearly one third of the world's human population and over 60 million people in the U.S. are estimated to be infected by the protozoan parasite Toxoplasma gondii. Although normally not life-threatening, this parasite can cause serious health problems and even death in people with weakened immune systems or in the developing fetus. Current drug therapies are unable to cure infection and long-term treatments often become toxic to the patient; the goal of this project is to identify essential parasite proteins responsible for establishing infection that have the capacity to serve as new potential therapeutic targets.

描述（由申请人提供）：弓形虫Gondii是一种广泛的原生动物寄生虫，能够导致发育中的婴儿和免疫功能低下的患者的严重并发症引起先天性疾病。近三分之一的人口长期感染了T. gondii。由于毒性，当前的疗法无法治愈慢性感染和不耐受，通常是由于长期治疗而引起的。我们有效治疗T. gondii感染的能力需要鉴定新的治疗靶标和制定合理的药物设计策略。 T. gondii在寄生众多的温血宿主方面非常有效，并且几乎可以感染任何成核细胞类型。在入侵过程中，T. gondii将来自分泌细胞器（称为Rhoptries）的蛋白质的异质混合物注入宿主细胞质中。结果是，宿主用来打击感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。涉及凋亡，代谢产物隔离和免疫反应的宿主途径有效地采用，使得贡迪t. gondii可以不受阻碍地完成其细胞内生命周期。 Rhoptry细胞器的蛋白质组学分析确定了高度扩展的丝氨酸/苏氨酸（S/T）激酶（ROP激酶）的家族（ROP激酶）以及对毒力基因的遗传学映射研究，涉及其中几个作为关键毒力因子。在T. gondii基因组和系统发育，结构和功能分析中已经确定了大约20种活跃的ROP激酶，表明它们与人类中的任何主要S/T激酶的主要系族都有显着差异。此后，几种多态性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，对于寄生虫的生存能力而言，没有一个是必不可少的。该应用的目的是通过系统地确定其在寄生虫的生存能力和毒力中的作用来对ROP激酶家族进行全面分析。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影响以及小鼠模型中的毒力。基本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进行比较来评估。天然核心ROP激酶复合物的隔离和宿主底物的捕获将通过提供有关可能在调节激酶功能或确定底物特异性方面作用的其他因素来完成分析。总而言之，该项目将整合ROP激酶复合物的各种表型分析，宿主细胞转录分析和蛋白质组学表征，从而对重要的ROP激酶在关键的宿主 - 病原体相互作用中起作用。更重要的是，由此产生的分析可以识别出许多新的潜在治疗靶标。 公共卫生相关性：据估计，近三分之一的世界人口和6000万人被原生动物的寄生虫弓形虫感染。尽管通常不会威胁生命，但这种寄生虫会导致严重的健康问题，甚至在免疫系统疲软或发育中的胎儿中死亡。当前的药物疗法无法治愈感染，长期治疗通常对患者有毒。该项目的目的是确定负责建立有能力作为新潜在治疗靶标的感染的基本寄生虫蛋白。