Functional characterization of Chlamydia trachomatis inclusion membrane proteins and their role in subversion of host vesicular trafficking

沙眼衣原体包涵膜蛋白的功能特征及其在破坏宿主囊泡运输中的作用

• 批准号: 10363758
• 负责人: Mary Weber
• 金额: $ 44.45万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10363758
• 关键词: Address; Animals; Antibiotics; Bacteria; Bacterial Infections; Bacterial Proteins; Binding; Blindness; Cell Culture Techniques; Cells; Ceramides; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Complex; Data; Defect; Development; Disease; Ectopic Pregnancy; Endosomes; Etiology; Genetic; Goals; Golgi Apparatus; Growth; Impairment; Incidence; Infection; Infertility; Integration Host Factors; Iron; Knowledge; Label; Lipids; Mediating; Membrane; Membrane Proteins; Metabolic; Microtubules; Molecular; Nutrient; Organelles; Paper; Pathogenesis; Pathway interactions; Pelvic Inflammatory Disease; Positioning Attribute; Prevalence; Process; Proteins; Recurrence; Recycling; Reporting; Role; SNAP receptor; Sexual Transmission; Sexually Transmitted Diseases; Source; Techniques; Therapeutic Intervention; Trachoma; Transferrin; Vacuole; Vesicle; Work; base; genetic manipulation; global health; insight; new therapeutic target; novel therapeutic intervention; pathogen; rab GTP-Binding Proteins; recruit; trafficking; trans-Golgi Network; vesicle transport

Project Summary Chlamydia trachomatis (C.t.) is the leading cause of non-congenital blindness and the most prevalent sexually transmitted bacterial infection in the world and thus, has a major impact on global health. C.t. replicates in a membrane-bound vacuole, termed the inclusion, that is extensively modified early in infection through the incorporation of over 50 type III secreted effector proteins termed inclusion membrane proteins (Incs). From within the confines of the inclusion, the bacterium must engage select host organelles to obtain key nutrients such as lipids and iron for bacterial replication and inclusion expansion. Despite the importance of nutrient acquisition to chlamydial infection, a major gap in our knowledge exists regarding how C.t. acquires these essential substrates from the host cell. Recent work from our group shows that the Inc protein CT229 binds and recruits Rab GTPases and specific Rab effectors to the inclusion. Using cutting-edge genetics, we showed that the absence of CT229 results in defects in intracellular replication and inclusion development. We hypothesize that CT229-Rab interactions direct post-Golgi vesicles and recycling endosomes to the inclusion for the acquisition of essential substrates needed for proliferation and inclusion expansion. In Aim 1, we will determine how CT229-Rab interactions promote lipid acquisition by hijacking host vesicular trafficking pathways and iron acquisition by promoting the recruitment of transferrin-positive vesicles to the inclusion. We will evaluate the lipid composition of the bacterial and inclusion membranes and evaluate the importance of lipid acquisition for incorporation into the inclusion and bacterial membranes. We will also answer a major long-standing question in the field by determining whether transferrin indeed serves as a major source of iron for chlamydia and furthermore mechanistically probe the mechanisms by which chlamydia may extract iron from this pathway. It is unlikely that CT229 alone controls vesicle hijacking from start to finish, therefore in Aim 2 we will identify the complex of host and bacterial proteins that are formed at the inclusion during CT229-Rab interactions. Using cellular, genetic, and molecular techniques we will evaluate the impact of Inc-Inc interactions on Rab-dependent vesicle transport to the inclusion. Detailed characterization of the bacterial and host proteins that co-opt host vesicular trafficking during C.t. infection will provide a holistic view of how intracellular pathogens coordinate the capture and fusion of host vesicles that are necessary for bacterial infection.

项目摘要 沙眼衣原体(Chlamydia trachomatis(C.T.)是非先天性失明的主要原因，也是最普遍的性 在世界上传播的细菌感染，因此，对全球健康有重大影响。C.T.在一个 膜结合的空泡，称为包涵体，在感染早期被广泛修饰，通过 被称为包涵膜蛋白(Incs)的50多种III型分泌效应蛋白的掺入。从… 在包涵体的范围内，细菌必须与选定的宿主细胞器接触以获得关键营养。 例如用于细菌复制和包裹体扩大的脂类和铁。尽管营养素很重要 对于衣原体感染的获得性感染，我们关于C.T.如何感染的知识存在一个主要缺口。获得了这些 来自宿主细胞的必需底物。我们小组最近的研究表明，Inc蛋白CT229结合并 招募Rab GTP酶和特定的Rab效应器加入。利用尖端遗传学，我们证明了 CT229的缺失导致细胞内复制和包涵体发育的缺陷。我们假设 CT229-Rab的相互作用将高尔基体后囊泡和循环内小体导向包涵体 获得增殖和扩大包涵体所需的基本底物。在目标1中，我们将确定 CT229-Rab相互作用如何通过劫持宿主囊泡转运途径和铁促进脂质获取 通过促进转铁蛋白阳性囊泡的招募而获得包涵体。我们会评估一下血脂 细菌膜和包涵体膜的组成以及评价脂质采集的重要性 被掺入包涵体和细菌膜中。我们还将回答一个长期存在的主要问题 通过确定转铁蛋白是否确实是衣原体和肺炎衣原体的主要铁源 此外，从机制上探索衣原体可能从这一途径中提取铁的机制。它是 CT229不太可能自始至终单独控制囊泡劫持，因此在目标2中，我们将确定 在CT229-Rab相互作用过程中在包涵体处形成的宿主和细菌蛋白的复合体。vbl.使用 细胞、遗传和分子技术我们将评估Inc.-Inc.相互作用对RAB依赖的影响 囊泡运输到包裹体。共选宿主的细菌和宿主蛋白的详细特征 CT过程中的囊泡运输。感染将提供细胞内病原体如何协调 捕获和融合细菌感染所必需的宿主小泡。