Dissecting the role of the Inclusion membrane protein IncE, a master multi-tasking scaffolding protein, in the pathogenesis of Chlamydia trachomatis infections

剖析包涵膜蛋白 IncE（一种主要的多任务支架蛋白）在沙眼衣原体感染发病机制中的作用

• 批准号: 10669588
• 负责人: Joanne N. Engel
• 金额: $ 67.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-22 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10669588
• 关键词: Affinity; Affinity Chromatography; Animal Model; Antibiotics; Bacteria; Binding; Biochemistry; Biological Assay; Biology; Cell membrane; Cells; Cellular biology; Chlamydia; Chlamydia trachomatis; Complement; Complex; Data; Data Set; Defect; Developing Countries; Disease; Early Endosome; Endosomes; Environment; Epitopes; Eye Infections; Family; Genetic; Grant; Human; In Vitro; Infection; Knowledge; Life Cycle Stages; Lipids; Liposomes; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Microbe; Microscopy; Mining; Modeling; Molecular; Multiprotein Complexes; Organelles; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Play; Positioning Attribute; Process; Production; Proteins; RNA Interference; Recurrence; Respiratory Tract Infections; Role; SNAP receptor; Scaffolding Protein; Structure; Testing; Transfection; Type III Secretion System Pathway; Vaccines; Variant; Vesicle; Virulence; cellubrevin; cost effective; experimental study; genital infection; human disease; induced pluripotent stem cell; insight; late endosome; live cell microscopy; mouse model; multitask; mutant; novel; obligate intracellular parasite; pathogen; peptidomimetics; protein complex; protein function; protein purification; protein transport; reproductive tract; scaffold; sorting nexins; syntaxin; trafficking

Abstract Chlamydia trachomatis (Ct) infections are important causes of human disease for which no vaccine exists. An important gap in our knowledge is how this obligate intracellular vacuolar bacterium establishes a privileged niche--a membrane bound compartment termed the inclusion. Chlamydia encode a distinctive family of secreted effectors, the Incs (Inclusion membrane proteins), which are translocated from the bacteria through the type III secretion system and inserted into the inclusion membrane. We have discovered that an early expressed Inc, IncE, binds to two different host cell protein complexes that are involved in trafficking, sorting nexins (SNX)5 and 6 as well as two closely related Q-SNARES, Syntaxin(STX)7 and STX12, which are involved in late and early endosome trafficking, respectively. Our results support a model wherein IncE directly binds SNX5/6, which redirects the ESCPE-1 complex (SNX5/6 together with SNX1/2) to the inclusion membrane, potentially disrupting ESCPE-1-mediated trafficking. In preliminary experiments, we have defined the regions of IncE that are necessary for binding to STX7/12 and discovered that IncE encodes an additional distinct short motif separate from the SNX5/6 cargo binding motif that mimics the zero layer motif (ZLM) of the VAMP3 family of R-SNARE proteins, known binding partners of STX7/12. This exciting observation suggests that IncE may modulate STX7/12 vesicle fusion with the inclusion, either by stimulating it, inhibiting it, or functioning as a tether. Using RNAi, we have discovered that STX7 and STX12 serve distinct non-overlapping roles in Ct infection. We have created an IncE null mutant and complemented it with informative IncE variants. This heroic application of Ct genetics will allow us to separate IncE binding to SNX5/6 from its binding to STX7/12, and to test the role of a single Inc in an animal model of Ct genital tract infection. In this grant, we propose to employ a combination of Ct genetics, cell biology, advanced microscopy, and biochemistry to: Aim 1. Determine the role of IncE in the pathogenesis of Ct infections. We will characterize the phenotypes of the IncE in-frame deletion mutant strain and informative isogenic complemented derivatives using (A) cell-based assays and (B) a mouse model of upper genital tract infection. Aim 2. Understand the molecular mechanism and consequences of IncE:STX7/12 interactions. Using informative IncE mutant strains and proteins, we will (A) determine whether IncE:STX7/12 interactions modulate fusion, using live cell microscopy that employs novel fluorescent lipid probes (B) determine if IncE, and specifically its ZLM, is sufficient to modulate STX7 vesicle fusion, using an in vitro liposome fusion assay; (C) test the hypothesis that the IncE ZLM binds directly to STX7 and/or STX12, using purified proteins; ad (D) Test whether IncE can bind simultaneously to STX7/12 and SNX5/6; Aim 3. Decode the roles of STX7/12 in regulating the Ct intracellular lifecycle. We will investigate (A) how STX12 contributes to homotypic inclusion fusion; (B) how STX7 contributes inclusion formation and production of infectious progeny. Our studies will increase our knowledge of host cell biology and reveal how microbes subvert these processes.

抽象的 沙眼衣原体（CT）感染是不存在疫苗的人类疾病的重要原因。 我们所知的一个重要差距是这种义务细胞内液泡细菌如何建立特权 利基 - 一种称为包容的膜结合室。衣原体编码一个独特的分泌家庭 效应子，Incs（包含膜蛋白），从细菌易位的III型 分泌系统并插入纳入膜。我们已经发现早期表达的公司， INCE，与参与运输的两个不同宿主细胞蛋白复合物结合，对Nexins（SNX）5和 6以及两个密切相关的Q-SNARES，语法（STX）7和STX12，涉及后期和早期 内体贩运。我们的结果支持一个模型，其中INCE直接绑定了SNX5/6，该模型 将ESCPE-1复合物（SNX5/6与SNX1/2一起）重定向到包含膜，可能会破坏 ESCPE-1介导的贩运。在初步实验中，我们定义了INCE的区域 与STX7/12结合所需的必要 从模拟R-SNARE的VAMP3家族的零层基序（ZLM）的SNX5/6货物结合图案 蛋白质，STX7/12的已知结合伴侣。这种令人兴奋的观察表明，INCE可能会调节 STX7/12囊泡融合通过刺激，抑制它或作为系绳起作用。使用 RNAi，我们发现STX7和STX12在CT感染中起着不同的非重叠作用。我们有 创建了一个INCE零突变体，并通过信息丰富的INCE变体进行了补充。 CT的英勇应用 遗传学将使我们能够将INCE与SNX5/6结合到与STX7/12的结合，并测试A的作用 CT生殖道感染动物模型中的单一INC。在这笔赠款中，我们建议采用 CT遗传学，细胞生物学，高级显微镜和生物化学至：AIM 1。确定INCE在 CT感染的发病机理。我们将表征INCE框内缺失突变体应变的表型 以及使用（a）基于细胞的测定和（b）上部的小鼠模型的信息源性辅助衍生物 生殖道感染。目标2。了解INCE的分子机制和后果：STX7/12 互动。使用信息丰富的INCE突变菌株和蛋白质，我们将（a）确定INCE是否：STX7/12 使用使用新型荧光脂质探针的活细胞显微镜调节融合（b）确定 如果INCE，尤其是其ZLM，足以使用体外脂质体融合来调节STX7囊泡融合 测定； （c）检验INCE ZLM使用纯化蛋白直接与STX7和/或STX12结合的假设； AD（D）测试INCE是否可以同时结合STX7/12和SNX5/6；目标3。解码角色 STX7/12在调节CT细胞内生命周期中。我们将调查（a）STX12如何贡献 同型包容融合； （b）STX7如何贡献纳入的形成和传染性后代的产生。 我们的研究将增加对宿主细胞生物学的了解，并揭示微生物如何颠覆这些过程。