TARGETING ESSENTIAL ROP KINASES IN TOXOPLASMA

描述(申请人提供)：弓形虫是一种广泛存在于野生、家养和同伴动物中的原生动物寄生虫，也通常感染人类。严重感染通常只在免疫功能低下的患者中发现，包括艾滋病毒感染、癌症化疗、器官移植或在宫内感染的婴儿。此外，新出现的研究表明，严重的眼弓形体病也可能发生在健康的成年人中，慢性感染是某些形式的精神疾病的潜在风险因素。弓形虫分离株在动物模型和人类感染中的毒力差异很大。最近的发现表明，这种寄生虫的主要毒力决定因素是来自棒状杆菌的分泌蛋白(ROP)，它们在入侵时被注入宿主细胞。许多ROP含有一个保守的丝氨酸/苏氨酸激酶结构域，并被预测为激酶，通过特定靶点的磷酸化潜在地改变宿主信号通路。在初步研究中，我们开发了一种系统性的基因干扰策略，以靶向基因组中所有已知的活性ROP激酶。在小鼠模型中，ROP激酶的破坏将被用来确定它们在体外和体内生长过程中的作用。另外，我们设计了一种单步插入策略来生成必需的ROP激酶的条件表达等位基因。单个ROPs的敲除将被用来探索由这些激酶控制的功能通路。我们还将使用生化分析和质谱学来鉴定ROP激酶的底物特异性，从而为抑制物检测的发展提供特定的底物。重组蛋白的表达将被用于开发体外活性分析和筛选有重点的激酶抑制物库以鉴定有效的抑制剂。这些研究的目的是为弓形虫中的必需激酶提供遗传学验证，并为未来的开发确定抑制ROP激酶的化学支架的特征。这些研究将有助于确定一类重要的寄生虫毒力因子的作用，并可能导致改进对弓形虫病的治疗干预。

DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread protozoan parasite of wild, domestic, and companion animals that also commonly infects humans. Severe infections are normally only found in immunocompromised patients, including HIV infection, cancer chemotherapy, organ transplant, or infants infected in utero. Additionally, emerging studies indicate that severe ocular toxoplasmosis can also occur in healthy adults, and that chronic infection is an underlying risk factor for some forms of psychiatric disease. Toxoplasma isolates differ dramatically in their virulence in animal models and also in human infections. Recent findings reveal that the major virulence determinants of this parasite are secretory proteins derived from the rhoptries (ROPs), which are injected to the host cell at the time of invasion. Many ROPs contain a conserved serine / threonine kinase domain and are predicted to function as kinases, potentially altering host signaling pathways by phosphorylation of specific targets. In preliminary studies, we have developed a systematic gene disruption strategy to target all of the known active ROP kinases in the genome. Disruption of ROP kinases will be used to define their roles during growth in vitro vs. in vivo in the mouse model. Separately, we have designed a single-step insertional strategy to generate conditional expression alleles of essential ROP kinases. Knockdown of individual ROPs will be used to explore the functional pathways controlled by these kinases. We will also employ biochemical assays and mass spectrometry to identify the substrate specificity of ROP kinases, there by providing specific substrates for development of inhibitor assays. Expression of recombinant proteins will be used to develop in vitro activity assays and to screen focused kinase inhibitor libraries to indentify potent inhibitors. The goal of these studies to provide genetic validation of essential kinases in T. gondii and to characterize chemical scaffolds that inhibitor ROP kinases for future development. These studies will help define the roles of an important class of parasite virulence factors and may lead to improved therapeutic intervention against toxoplasmosis.