Structural insight into novel mechanisms of type III secretion

III 型分泌新机制的结构洞察

• 批准号: 10609868
• 负责人: CHARALAMPOS KALODIMOS
• 金额: $ 44.88万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609868
• 关键词: ATP phosphohydrolase; Animals; Anti-Infective Agents; Bacterial Proteins; Binding; Biochemical; Biological Assay; Bordetella; Cell Surface Extensions; Cells; Child Mortality; Classification; Complex; Cryoelectron Microscopy; Cytosol; Data; Dedications; Disease; Dysentery; Event; Filament; Food; Food Poisoning; Gram-Negative Bacteria; Human; In Vitro; Infantile Diarrhea; Kinetics; Light; Locomotion; Membrane; Molecular Chaperones; Morphology; Needles; Organelles; Pathogenesis; Pathogenicity; Plague; Process; Protein Export Pathway; Protein Secretion; Proteins; Proton-Motive Force; Pseudomonas; Recycling; Regulation; Reporting; Resolution; Role; Salmonella; Shigella; Signal Transduction; Sorting; Specificity; Structure; System; Thermodynamics; Time; Type III Secretion System Pathway; Typhoid Fever; Vaccines; Virulence; Virulence Factors; X-Ray Crystallography; cell motility; design; enteropathogenic Escherichia coli; in vivo; insight; kinetosome; nanomachine; novel; operation; pathogenic Escherichia coli; pathogenic bacteria; protein structure; structural biology; therapeutic protein

Project Summary Type III secretion systems (T3SSs) are nanomachines that are dedicated to protein export in Gram-negative bacteria. T3SSs share the same morphology and overall structure and can be functionally classified into two evolutionary-related classes: the flagellar T3SS, which promotes bacterial locomotion and motility enabled by the flagellum, and the pathogenic T3SS, which uses the injectisome to transport virulence proteins into human or animal host cells. Over the past decade significant progress has been made in understanding the structure, assembly and the mode of operation of T3SS. The principal structural building proteins of the flagellum and the injectisome, from the basal body embedded in the inner and outer bacterial membrane to the tip of the filaments protruding from the cell surface, and the cytosolic components have been extensively characterized. Flagellar proteins and virulence factors (effectors, needle proteins and translocators) form tight complexes with T3S-dedicated chaperones in the cytosol and are subsequently targeted specifically to the export apparatus located at the membrane. Powered by ATP and the proton motive force, the flagellar proteins and bacterial effectors are then translocated through the channel. Fundamental questions about the functional mechanisms underpinning these processes remain unaddressed. We propose to use an integrated approach combining structural, dynamic, thermodynamic, kinetic, biochemical and in vitro and in vivo functional assays to provide insight into the early events of the translocation process that involve the recognition mechanisms by chaperones, targeting mechanisms to the ATPase and the sorting platform, selection mechanisms that control the hierarchical transport of the filament-forming proteins and the effectors and ultimately the assembly of the cytosolic part of the machinery. We have extensively characterized over the last years T3S protein components from the enteropathogenic Escherichia coli (EPEC), the major cause of infantile diarrhea and child mortality worldwide, as well as from Salmonella sp. commonly associated with food poising. We present here novel findings supporting very intriguing hypotheses about the mechanisms used by T3SSs to carry out their function. The specific aims are designed to provide atomic-resolution insight into (i) the mechanisms of specific interactions between and among key T3S proteins, (ii) the mechanistic basis for targeting of T3S proteins to the export gate, (iii) the “recognition” and “secretion” signal, and (iv) the assembly and operation mechanisms of the export gate and ultimately of the entire T3S machinery.

项目摘要 III型分泌系统（T3SSS）是纳米机器，用于革兰氏阴性细菌中蛋白质输出。 T3SS共享相同的形态和整体结构，并且可以在功能上经典地分为两个进化相关 课程：拟南芥的动力和运动能力，并由致病性促进了细菌的运动和运动性，该类别的植物T3SS。 T3SS，它使用注射剂将病毒蛋白运输到人或动物宿主细胞中。 在过去的十年中，在理解结构，组装和模式方面取得了重大进展 T3SS的操作。基本体的主要结构建筑蛋白和注射体的蛋白质 嵌入内部和外细菌膜中 胞质成分已被广泛表征。鞭毛蛋白和病毒因子（效应子，针头 蛋白质和易位剂）与细胞质中的T3S划分的伴侣形成紧密的复合物，随后进行 专门针对位于膜上的出口设备。由ATP和质子动力驱动， 然后通过通道易位，叶片蛋白和细菌效应。关于 支撑这些过程的功能机制仍未得到解决。 我们建议使用结合结构，动态，热力学，动力学，生化和中的集成方法 体外和体内功能分析，以洞悉涉及易位过程的早期事件 伴侣的识别机制，针对ATPase的靶向机制和排序平台，选择 控制形成蛋白质的分层运输的机制以及作用以及最终 机械胞质部分的组装。在过去的几年中，我们已经广泛表征了T3S蛋白 肠病大肠杆菌（EPEC）的成分，婴儿腹泻和儿童死亡率的主要原因 全球以及Salmonella sp。通常与食物中毒有关。我们在这里提出了支持 关于T3SS用于执行其功能的机制的非常有趣的假设。具体目的是 旨在提供对（i）关键T3之间特定相互作用的机制的原子分辨率洞察力 蛋白质，（ii）将T3S蛋白靶向出口门的机械基础，（iii）“识别”和“分泌” 信号，以及（iv）导出门的组装和操作机理以及整个T3S机械的最终。