Structure-function relationships of Chlamydia trachomatis Tarp & binding proteins

描述（由申请人提供）：专性细胞内细菌沙眼衣原体在美国和世界范围内引起大量发病率。由于无法操纵衣原体基因组，因此缺乏真正的毒力因子。因此，衣原体感染的致病机制描述不多，需要进一步研究。梭沙眼衣原体的一种效应因子，称为Tarp，是一种候选的毒力因子。Tarp被宿主细胞激酶酪氨酸磷酸化，并与衣原体进入过程中的肌动蛋白募集有关。已经鉴定了Tarp蛋白结构域，其包括：i）肌动蛋白结合和成核结构域，ii）富含脯氨酸的寡聚化结构域和iii）磷酸化结构域。Tarp磷酸化结构域的功能目前尚不清楚，也是所附资助提案的重点。含Src同源结构域2（SH 2）的蛋白是候选的Tarp相互作用蛋白。这项资助提案的目的是通过使用检测蛋白质-蛋白质相互作用的测定法（如GST融合下拉测定法、免疫沉淀法和两种旨在靶向含SH 2结构域的蛋白质的市售测定法）来鉴定Tarp结合蛋白（具有和不具有SH 2结构域的蛋白质）。第二，评估衣原体进入和发展中对已鉴定的Tarp结合蛋白的任何需求。最近，PIS激酶与磷酸化Tarp有关。通过PI 3 K修饰脂质最终调节多种细胞过程。我们预测，Tarp招募PI 3 K在衣原体感染的建立中起着至关重要的作用，并打算研究这种关联与化学抑制剂和干扰RNA的要求。目前还没有衣原体疫苗。阐明Tarp与宿主的相互作用对于绘制C.沙眼衣原体在感染细胞内建立居所。更好地了解衣原体启动成功感染的机制将导致新的治疗干预措施的确定。 相关性：去年仅在美国，就有100多万人感染了性传播疾病沙眼衣原体。最近发现的一种称为Tarp的衣原体蛋白被认为在感染的初始阶段起着重要作用。这项研究计划的目标是确定Tarp蛋白如何促进衣原体引起疾病。

DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis causes substantial morbidity in the US and world wide. An inability to manipulate the chlamydial genome has consequently produced a dearth of bona fide virulence factors. Hence, pathogenic mechanisms of chlamydial infections are poorly described and require further examination. A C. trachomatis effector called Tarp, for translocated actin recruiting protein is a candidate virulence factor. Tarp is tyrosine phosphorylated by a host cell kinase and is associated with actin recruitment during Chlamydiae entry. Tarp protein domains have been identified and include: i) an actin binding and nucleating domain, ii) a proline rich oligomerization domain and iii) a phosphorylation domain. The function of the Tarp phosphorylation domain is currently unknown and the focus of the attached grant proposal. Src-homology domain 2 (SH2) containing proteins are candidate Tarp interacting proteins. The purpose of this grant proposal is to identify Tarp binding proteins (those with and without SH2 domains) by using assays to detect protein-protein interactions such as GST-fusion pull-down assays, immunoprecipitation, and two commercially available assays designed to target SH2 domain containing proteins. And secondly, to assess any requirements for identified Tarp binding proteins in Chlamydiae entry and development. Recently, PIS kinase was implicated in association with phosphorylated Tarp. Modification of lipids by PI3K ultimately regulates a variety of cellular processes. We predict that Tarp recruitment of PI3K plays a vital role in the establishment of chlamydial infection and intend to examine a requirement for this association with chemical inhibits and interfering RNAs. Currently, a chlamydial vaccine is not available. Elucidation of Tarp interactions with the host is crucial for mapping signaling pathways used by C. trachomatis to establish residence within infected cells. A better understanding of the mechanisms employed by Chlamydiae to initiate a successful infection will result in the identification of novel therapeutic interventions. RELEVANCE: The sexually transmitted disease causing bacteria, Chlamydia trachomatis, infected more than 1 million people in the US alone last year. A recently identified Chlamydiae protein called Tarp is believed to play an important role in the initial stages of infection. The goal of this research proposal is to determine how the Tarp protein facilitates Chlamydiae to cause disease.