Structural insight into novel mechanisms of type III secretion

III 型分泌新机制的结构洞察

• 批准号: 8775192
• 负责人: CHARALAMPOS KALODIMOS
• 金额: $ 37.64万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8775192
• 关键词: ATP phosphohydrolase; Adopted; Animals; Anti-Infective Agents; Bacteria; Binding; Biochemical; Biological Assay; Bordetella; Cell physiology; Cell surface; Cells; Child Mortality; Complex; Cytoplasm; Cytosol; Data; Defect; Disease; Dysentery; Eukaryotic Cell; Event; Food Poisoning; Human; In Vitro; Infantile Diarrhea; Infection; Insecta; Kinetics; Membrane; Molecular Chaperones; Molecular Conformation; Needles; Nematoda; Nucleotides; Pathogenesis; Plague; Plants; Preparation; Process; Property; Protein Export Pathway; Proteins; Proton-Motive Force; Pseudomonas; Resolution; Role; Salmonella; Shigella; Signal Transduction; Spottings; Structure; System; Thermodynamics; Type III Secretion System Pathway; Typhoid Fever; Vaccines; Virulence; Virulence Factors; base; biological systems; biophysical analysis; design; enteropathogenic Escherichia coli; in vivo; insight; kinetosome; mimicry; nanomachine; novel; operation; pathogen; pathogenic Escherichia coli; pathogenic bacteria; prevent; prototype; secretion process; success; therapeutic protein

DESCRIPTION (provided by applicant): A large number of bacterial pathogens, including Shigella, Salmonella, Bordetella, Pseudomonas, and pathogenic E. coli that are pathogenic for humans, other animals including insects or nematodes, and plants are equipped with a special protein- export apparatus called a type III secretion system (TTSS) or an injectisome. The injectisome is a highly sophisticated nanomachine that has specifically evolved to allow bacteria to deliver proteins into eukaryotic cells. The TTSS enables these pathogens to inject virulence proteins (known as effectors) directly into the cytoplasm of the eukaryotic host cells they infect. Many of these type III translocated effectors mimic eukaryotic factors and are capable of subverting key host cellular processes to the benefit of the pathogen during infection. Over the past decade, significant progress has been made in understanding the structure, assembly and the mode of operation of TTSS. The cytosolic components, the principal structural building proteins of the injectisome, from the basal body embedded in the inner and outer bacterial membrane to the tip of the needle protruding from the cell surface, have been extensively characterized. Virulence factors (effectors, needle proteins and translocators) form tight complexes with cognate chaperones in the cytosol and are subsequently targeted specifically to an ATPase protein located at the base of the injectisome. Powered by ATP, the effector is then translocated through the needle and is secreted in the eukaryotic cell. 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 secretion process that involve the recognition and binding of virulence factors (effectors and translocators) by cognate chaperones and the targeting of these substrates to the ATPase. We have extensively characterized over the last 3 years TTS protein components from the enteropathogenic Escherichia coli (EPEC), a prototype for TTSS and the major cause of infantile diarrhea and child mortality worldwide. The specific aims are designed to provide atomic-resolution insight into (i) the mechanisms of specific interaction between TTS chaperones and virulence factors, (ii) the structural and dynamic properties of the ATPase, (iii) the "recognition" or "secretion" signal, and (iv) the specific targeting of chaperone-substrate complexes to the ATPase.

DESCRIPTION (provided by applicant): A large number of bacterial pathogens, including Shigella, Salmonella, Bordetella, Pseudomonas, and pathogenic E. coli that are pathogenic for humans, other animals including insects or nematodes, and plants are equipped with a special protein- export apparatus called a type III secretion system (TTSS) or an injectisome.注射体是一种高度复杂的纳米机械，已专门演变为允许细菌将蛋白质输送到真核细胞中。 TTSS使这些病原体能够将毒力蛋白（称为效应子）直接注入其感染的真核宿主细胞的细胞质中。这些型III型易位效应子中的许多模仿真核因子，并且能够颠覆关键的宿主细胞过程，从而在感染过程中受益于病原体。在过去的十年中，在理解TTSS的结构，组装和操作方式方面取得了重大进展。胞质成分是注射体的主要结构建筑蛋白，从嵌入内部和外细菌膜的基体到从细胞表面突出的针头的尖端，已经广泛表征。毒力因子（效应子，针蛋白和转运剂）与细胞质中的同源伴侣形成紧密的复合物，随后专门针对位于注射体底部的ATPase蛋白。然后由ATP提供动力，然后通过针头易位，并在真核细胞中分泌。有关这些过程支撑的功能机制的基本问题仍然没有解决。我们建议使用一种集成方法，结合结构，动态，热力学，动力学，生化和体外和体内功能测定法，以洞悉分泌过程的早期事件，这些事件涉及毒力因子（效应者和转运剂）的识别和结合，通过认真的伴侣和这些substrate substrate of spase to aTpase topase aTpase aTpase aTpase。在过去的3年中，我们广泛表征了来自肠病毒性大肠杆菌（EPEC）的TTS蛋白成分，这是TTSS的原型，以及全世界婴儿腹泻和儿童死亡率的主要原因。该特定目的旨在提供（i）TTS伴侣和毒力因子之间特定相互作用的机制，（ii）ATPase的结构和动态特性，（iii）“识别”或“分泌”信号，以及（iv）冠层蛋白酶对Atpase的特定靶向。