Molecular dissection of the Chlamydia trachomatis Tarp effector in vivo

 描述(由申请人提供)：专性细胞内沙眼衣原体细菌在美国和世界范围内导致大量发病率。沙眼衣原体的一种名为Tarp的转位肌动蛋白招募蛋白是一种候选毒力因子。TARP是一种被宿主细胞激酶磷酸化的酪氨酸，与沙眼衣原体进入时的肌动蛋白募集有关。到目前为止，所有的衣原体参考和临床分离株都含有TARP基因。我们已经确定了四个Tarp蛋白结构域，包括：i)一个磷酸化结构域；ii)一个富含脯氨酸的寡聚化结构域；iii)一个G-肌动蛋白结合和核化结构域；以及iv)两个F-肌动蛋白结合和捆绑结构域。我们假设特定的Tarp结构域是细菌进入和/或衣原体发育所必需的。我们将通过以下方式验证这一假设：1)分析表达TARP结构域缺失突变体的沙眼衣原体转化体，并检查突变基因作为显性负等位基因的能力，从而影响宿主上皮细胞中细菌克隆的入侵和发育；2)使用独特的遗传策略来针对携带野生型或突变型TARP质粒副本的沙眼衣原体转化体中TARP基因的染色体拷贝的缺失。阐明沙眼衣原体启动成功感染的分子机制可能提供线索，可应用于这种多产病原体的新的治疗干预措施。

 DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis causes substantial morbidity in the US and worldwide. 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 C. trachomatis entry. All reference and clinical isolates of Chlamydiae species examined to date harbor the tarP gene. We have identified and biochemically characterized four Tarp proteins domains including: i) a phosphorylation domain ii) a proline rich oligomerization domain iii) a G-actin binding and nucleating domain, and iv) two F-actin binding and bundling domains. We hypothesize that specific Tarp domains are required for bacterial entry and/or chlamydial development. We will test this hypothesis by: 1) analyzing C. trachomatis transformants that express Tarp domain deletion mutants and examine the ability of the mutant genes to function as dominant negative alleles thereby affecting invasion and development of the bacterial clones in host epithelial cells and 2) using a unique genetic strategy to target deletion of the chromosomal copy of the tarP gene in C. trachomatis transformants carrying a plasmid copy of wild type or mutant tarP. Elucidation of the molecular mechanisms employed by C. trachomatis to initiate a successful infection may provide clues that can be applied to novel therapeutic interventions for this prolific pathogen.