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: 10411625
• 负责人: Mary Weber
• 金额: $ 8.09万
• 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 2022-08-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10411625
• 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.

项目概要 沙眼衣原体 (C.t.) 是非先天性失明的主要原因，也是最常见的性病 细菌感染在世界范围内传播，从而对全球健康产生重大影响。 C.t.复制在一个 膜结合液泡，称为包涵体，在感染早期通过 掺入超过 50 种 III 型分泌效应蛋白，称为包涵膜蛋白 (Incs)。从 在包容性的范围内，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 例如用于细菌复制和内含物扩张的脂质和铁。尽管营养很重要 衣原体感染的获得性，我们对于 C.t. 如何获得衣原体感染的知识存在重大差距。获得这些 来自宿主细胞的必需底物。我们小组最近的工作表明 Inc 蛋白 CT229 结合并 招募 Rab GTPases 和特定的 Rab 效应子纳入其中。利用尖端遗传学，我们证明了 CT229 的缺失会导致细胞内复制和包涵体发育缺陷。我们假设 CT229-Rab 相互作用指导高尔基体后囊泡和循环内体以包含 获取增殖和包容性扩展所需的基本底物。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运输途径和铁来促进脂质获取 通过促进转铁蛋白阳性囊泡募集到包涵体来获得。我们将评估血脂 细菌和包涵体膜的组成并评估脂质获取的重要性 掺入包涵体和细菌膜。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是衣原体铁的主要来源和 此外，还从机制上探讨了衣原体从该途径中提取铁的机制。这是 CT229 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 CT229-Rab 相互作用过程中包涵体形成的宿主和细菌蛋白复合物。使用 细胞、遗传和分子技术，我们将评估 Inc-Inc 相互作用对 Rab 依赖性的影响 囊泡运输至包涵体。共同选择宿主的细菌和宿主蛋白的详细表征 C.t 期间的囊泡运输感染将提供细胞内病原体如何协调的整体视图 捕获和融合细菌感染所必需的宿主囊泡。