A molecular switch of the type III secretion system in Chlamydia trachomatis

描述(申请人提供)：沙眼衣原体III型分泌系统的分子开关沙眼衣原体是世界上最常见的性传播疾病的病原体，是严重的公共卫生负担。女性生殖器衣原体感染的严重后遗症包括盆腔炎、宫外孕和输卵管不孕。沙眼衣原体的一个关键毒力机制是III型分泌系统(T3SS)，它直接将蛋白效应物运送到宿主细胞胞浆中，破坏宿主免疫，使细菌能够在宿主体内生存。本项目的目的是研究沙眼衣原体控制III型分泌物和基因表达的机制，从而促进疾病的发病。我们推测，在沙眼衣原体感染过程中，T3SS活性是通过CT663及其蛋白伴侣组成的分子开关来调节的，并与基因转录相偶联。我们的新发现有力地支持了这一假说，即衣原体CT663是一个双功能蛋白，既是一个转录调节蛋白，与含有？66的RNA聚合酶相互作用，又是COPN的T3SS伴侣，它既是T3SS的调节因子，也是T3SS的效应器。我们的具体目标是：目的1.检验CT663形成III型分泌活性所必需的蛋白质复合体的假设。我们将使用蛋白质-蛋白质相互作用、免疫检测和致病性大肠杆菌(EPEC)系统的定量分析来表征CT663及其合作伙伴的动态相互作用，以及这些相互作用如何影响分泌活性。目的2.验证CT663差异调控基因转录的假说。这一目标将 使用我们建立的重组？66RNA聚合酶在体外和在大肠杆菌中的转录检测来实现。这些研究将揭示T3SS活性如何影响沙眼衣原体感染期间的基因表达，反之亦然。该项目将通过协调T3SS的调节事件和感染期间的基因表达，为沙眼衣原体如何利用T3SS在细胞内生态位中生存提供重要的见解。我们的研究将极大地扩展目前对沙眼衣原体感染过程的了解，并有助于确定潜在的药物靶点，用于新的治疗方法，从而显著减轻沙眼衣原体感染造成的公共卫生负担。

DESCRIPTION (provided by applicant): A molecular switch of the type III secretion system in Chlamydia trachomatis The obligate intracellular bacterium C. trachomatis is the causative agent of the most common sexually transmitted disease worldwide and represents a significant public health burden. The severe sequelae of genital chlamydial infections in women include pelvic inflammatory disease, ectopic pregnancy and tubal infertility. A key virulence mechanism of C. trachomatis is the type III secretion system (T3SS) that directly delivers protein effectors into the host cell cytosol to subvert host immunity and enables bacterial survival in hosts. The objective of this project is to study the mechanism by which C. trachomatis controls type III secretion and gene expression, contributing to disease pathogenesis. We hypothesize that T3SS activity is regulated and coupled to gene transcription during C. trachomatis infection by a molecular switch, consisting of CT663 and its protein partners. This hypothesis is strongly supported by our novel finding that chlamydial CT663 is a bi-functional protein acting as both a transcription regulator that interacts with RNA polymerase containing ? 66 and a T3SS chaperone for CopN, which is a regulator as well as an effector of the T3SS. Our specific aims are: Aim 1. To test the hypothesis that CT663 forms a protein complex necessary for type III secretion activity. We will characterize the dynamic interactions of CT663 and its partners, and how these interactions impact the secretion activity using quantitative assays for protein-protein interactions, immunodetection, and an enteropathogenic Escherichia coli (EPEC) system. Aim 2. To test the hypothesis that CT663 differentially regulates the gene transcription. This aim will be achieved using our established transcription assays with reconstituted ?66RNA polymerase in vitro and in E. coli. These studies will uncover how T3SS activity affects gene expression during C. trachomatis infection and vice versa. This project will provide important insights into how C. trachomatis utilizes T3SS to survive in an intracellular niche by coordinating the regulatory events of the T3SS and gene expression during infection. Our research will significantly expand current knowledge of the C. trachomatis infection process and contribute to the identification of potential drug targets for new therapies that could significantly reduce the public health burden caused by C. trachomatis infection.