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: 10596075
• 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/10596075
• 关键词: Address; Animal Model; Antibiotic Therapy; 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; Metabolism; Microtubules; Molecular; Nutrient; Nutritional Requirements; Organelles; Paper; Pathogenesis; Pathway interactions; Pelvic Inflammatory Disease; Positioning Attribute; Prevalence; Process; Proliferating; Proteins; Recurrence; Recycling; Reporting; Role; SNAP receptor; Sexual Transmission; Sexually Transmitted Diseases; Source; Techniques; Therapeutic Intervention; Trachoma; Transferrin; Vacuole; Vesicle; Work; 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（C.t.）是导致非先天性失明的主要原因， 在世界上传播的细菌感染，因此，对全球健康产生重大影响。C.T.复制在 膜结合的空泡，称为包涵体，在感染早期通过 超过50种III型分泌效应蛋白的掺入，称为包涵体膜蛋白（Incs）。从 在内含物的范围内，细菌必须与选定的宿主细胞器结合，以获得关键的营养物质 例如脂质和铁，用于细菌复制和内含物扩增。尽管营养的重要性 获得衣原体感染，在我们的知识中存在一个主要的差距，关于如何C.获取这些 从宿主细胞中提取必需底物。我们小组最近的工作表明，Inc蛋白CT 229结合， 招募Rab GTP酶和特异性Rab效应物。利用最先进的遗传学，我们发现， CT 229的缺失导致细胞内复制和包涵体发育的缺陷。我们假设 CT 229-Rab相互作用引导高尔基体后囊泡和再循环内体进入内含物， 获得增殖和包含物扩增所需的基本底物。在目标1中，我们将确定 CT 229-Rab相互作用如何通过劫持宿主囊泡运输途径和铁促进脂质获得 通过促进转铁蛋白阳性囊泡向内含物的募集来获得。我们将评估脂质 细菌和包涵体膜的组成，并评估脂质获取对 掺入到包涵体和细菌膜中。我们还将回答一个长期存在的重大问题， 通过确定转铁蛋白是否确实作为衣原体的主要铁来源， 此外，机械地探测衣原体可从该途径提取铁的机制。是 CT 229不太可能从头到尾单独控制囊泡劫持，因此在目标2中，我们将确定 在CT 229-Rab相互作用期间在包涵体处形成的宿主和细菌蛋白质的复合物。使用 细胞，遗传和分子技术，我们将评估Inc-Inc相互作用对Rab依赖性 囊泡运输到内含物。细菌和宿主蛋白的详细表征， 在C.t.感染将提供细胞内病原体如何协调 细菌感染所必需的宿主囊泡的捕获和融合。