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，因此造成了真正的毒力因子的缺乏。因此，衣原体感染的致病机制尚不清楚，需要进一步研究。一种被称为Tarp的沙眼衣原体效应物（易位肌动蛋白招募蛋白）是一种候选毒力因子。Tarp是被宿主细胞激酶磷酸化的酪氨酸，在衣原体进入时与肌动蛋白募集有关。已确定的Tarp蛋白结构域包括：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，这是所附拨款提案的重点。含有Src-homology domain 2 （SH2）的蛋白是Tarp相互作用的候选蛋白。该拨款提案的目的是通过检测蛋白-蛋白相互作用（如gst融合下拉试验、免疫沉淀和两种市售的针对含有SH2结构域的蛋白的试验）来鉴定Tarp结合蛋白（具有和不具有SH2结构域的蛋白）。其次，评估衣原体进入和发育过程中对已鉴定的Tarp结合蛋白的需求。最近，PIS激酶与磷酸化的Tarp有关。通过PI3K修饰脂质最终调节多种细胞过程。我们预测，PI3K的Tarp募集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和干扰rna的需求。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沙眼衣原体在感染细胞内建立居所的信号通路至关重要。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。

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.