Functional characterization of early Chlamydia effectors

项目说明/摘要/摘要 沙眼衣原体是一种广泛传播的专性胞内细菌。 感染泌尿生殖道上皮表面的病原体，导致严重的后遗症，如盆腔 炎症性疾病和不孕不育。在感染的最初阶段，衣原体可能会 10-15个单独的III型(T3s)“效应”蛋白分泌到目标宿主细胞中。我们 假设这些衣原体效应蛋白协同工作，在时间上重新编程 信号通路和细胞骨架功能促进细胞侵袭和建立新生 致病空泡(“包涵体”) 在我们的第一个目标中，我们建议将新出现的遗传和分子遗传学工具应用于衣原体 明确早期效应物在侵袭、包涵体成熟和体内感染中所起的作用。我们也 建议通过以下方式对效应器突变体组合进行上位性分析 功能基团，并优先考虑作为核心的效应器的详细分子特征 信令节点。 在我们的第二个目标中，我们将重点放在Tepp的特征上，Tepp是一个扮演重要角色的效应器 对信号通路进行重新编程，包括参与先天免疫反应的通路。我们建议 确定I类磷脂酰肌醇3激酶(PI3K)在新生阶段的激活机制 包裹体及其活性在膜动力学调节和激活中的作用 I型干扰素反应。同时，我们将确定导致TEPP的分子参与者- 介导的激活和修饰Eps8，Rac1活性和细胞-细胞连接的调节因子，通过 应用“邻近蛋白质组学”方法。 总体而言，我们的研究计划寻求执行系统层面的功能评估 早期效应器和选定效应器作用机制的详细分子特征。 总的目标是定义特定效应器(S)如何发挥作用的分子基础，确定如何 信号通路被重新编程，以及它们最终如何对衣原体在 宿主组织和病理的诱导。

Project Description/Summary/Abstract The obligate intracellular bacterium Chlamydia trachomatis is a widely disseminated obligate pathogen that infects epithelial surfaces of the urogenital tract, leading to severe sequela such as pelvic inflammatory disease, and infertility. During the initial stages of infection Chlamydia likely delivers between 10-15 separate Type III secreted (T3S) “effector” proteins into the target host cell. We hypothesize that these Chlamydia effector proteins work co-operatively to temporally reprogram signaling pathways and cytoskeletal functions to promote cell invasion and establish a nascent pathogenic vacuole (“inclusion”) In our first aim, we propose to apply emerging genetic and molecular genetic tools in Chlamydia to define the role of early effectors play in invasion, inclusion maturation and in vivo infections. We also propose to perform an epistatic analysis of combinations of effector mutants to group effector by functional groups and to prioritize a detailed molecular characterization of effector that are central signaling nodes. In our second aim, we focus on the characterization of Tepp, an effector that plays important roles in reprograming signaling pathways, including those involved in innate immune responses. We propose to define the mechanism of activation of Class I phosphoinositide 3 kinases (PI3K) at nascent inclusions and the role these activities play in the regulation of membrane dynamics and activation of Type I interferon responses. In parallel, we will identify the molecular players that lead to the Tepp- mediated activation and modification of Eps8, a regulator of Rac1 activity and cell-cell junctions, by applying “proximity proteomics” approaches. Overall, our research plan seeks to perform both a systems levels assessment of the function of early effectors and a detailed molecular characterization of mechanism of action for selected effectors. The overall goal is to define the molecular basis of how specific effector(s) function, identify how signaling pathways are re-programmed and how they all ultimately contribute to Chlamydia survival in host tissues and the induction of pathology.