A Mechanism for Shigella Type III Secretion Activation

III 型志贺氏菌分泌激活机制

• 批准号: 8071514
• 负责人: WILLIAM D. PICKING
• 金额: $ 17.82万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8071514
• 关键词: Affect; Antigens; Bacillary Dysentery; Bacteria; Behavior; Binding; Binding Sites; Biological Assay; Biological Models; Biophysics; Cells; Cholesterol; Complex; Data; Dissection; Docking; Epithelial Cells; Future; Hemolysis; Human; Incubated; Individual; Infection; Injection of therapeutic agent; Intestines; Investigation; Ligands; Liposomes; Mediating; Membrane; Methods; Molecular; Mutation; Needles; One-Step dentin bonding system; Pathogenesis; Pathway interactions; Plasmids; Positioning Attribute; Process; Property; Protein Biochemistry; Proteins; Public Health; Recruitment Activity; Role; Shigella; Shigella Infections; Shigella flexneri; Signal Transduction; Site; Solutions; Sphingomyelins; Structure; Surface; System; Testing; Type III Secretion System Pathway; Virulence; base; deoxycholate; design; human disease; kinetosome; novel; pathogen; polymerization; prevent; protein folding; public health relevance; structural biology; tool; trait

DESCRIPTION (provided by applicant): Shigella flexneri, the causative agent of bacillary dysentery, uses its type III secretion system (TTSS) to deliver proteins into host cells to promote bacterial entry. From its position at the tip of the TTSS needle, invasion plasmid antigen D (IpaD) serves to control Shigella type III secretion. By incubating Shigella with deoxycholate (DOC), we have shown that the first secreted translocator protein, IpaB, can be recruited to the needle tip complex without further induction of type III secretion. Recruitment of the second translocator (IpaC) to the needle tip complex occurs upon incubating the bacteria with liposomes rich in cholesterol and sphingomyelin, which also results in full induction of type III secretion. Thus, the Shigella TTSS provides a novel model system for exploring the individual steps of type III secretion induction. Based on the preliminary findings presented here, we hypothesize that IpaD senses environmental signals to trigger the controlled recruitment of IpaB to the needle tip complex. Because DOC mimics the environmental signal(s) needed for this distinct step on the pathway to type III secretion induction, we plan to determine the physical and molecular mechanism responsible for IpaB recruitment to the TTSA needle tip complex. To do this, the specific aims of this investigation are to: 1) solve the crystal structure of IpaD with DOC bound to identify the conformational changes responsible for IpaB recruitment to the Shigella surface; 2) target key sites on IpaD for mutational analysis and phenotypic characterization; and 3) determine the influence of targeted mutations on solution properties of IpaD IpaD and generate new crystals and co-crystals of IpaD 1 DOC to determine how specific mutations that alter IpaD function affect its structure and dynamics. TTSSs are essential virulence determinants for many significant human pathogens. Using the Shigella system, we are the first to describe the distinct steps of type III secretion induction. This unprecedented dissection of the steps of type III secretion now provides a tool for revealing the structural basis for type III secretion induction. In this investigation, we have embarked on a collaborative effort to explore type III secretion at a level not currently possible in any other bacterial system. The information obtained here will contribute to the future design of practical methods for blocking type III secretion. PUBLIC HEALTH RELEVANCE: Shigella flexneri is global public health problem as the causative agent of bacillary dysentery (shigellosis). To initiate infection, S. flexneri uses a complex type III secretion system to deliver proteins into human intestinal epithelial cells to promote bacterial entry into these cells. In this investigation, we will identify the mechanism by which this secretion system becomes activated so that it may be possible to identify compounds that can neutralize Shigella's ability to cause human disease.

描述（由申请人提供）：福氏志贺氏菌是细菌性痢疾的病原体，使用其III型分泌系统（TTSS）将蛋白质传递到宿主细胞以促进细菌进入。侵袭质粒抗原D （IpaD）位于TTSS针尖，可控制志贺氏菌III型的分泌。通过用脱氧胆酸盐（DOC）培养志贺氏菌，我们发现第一个分泌的转运蛋白IpaB可以被招募到针尖复合体中，而无需进一步诱导III型分泌。当细菌与富含胆固醇和鞘磷脂的脂质体孵育时，第二转运体（IpaC）向针尖复合体募集，这也导致III型分泌的充分诱导。因此，志贺氏菌TTSS为探索III型分泌诱导的各个步骤提供了一个新的模型系统。基于本文提出的初步研究结果，我们假设IpaD感知环境信号触发IpaB到针尖复合体的受控招募。由于DOC模拟了诱导III型分泌通路中这一独特步骤所需的环境信号，我们计划确定IpaB募集到TTSA针尖复合体的物理和分子机制。为此，本研究的具体目的是：1)利用DOC结合求解IpaD的晶体结构，以确定IpaB向志贺氏菌表面募集的构象变化；2)针对IpaD上的关键位点进行突变分析和表型鉴定；3)确定靶向突变对IpaD IpaD溶液性质的影响，生成IpaD 1 DOC的新晶体和共晶体，以确定改变IpaD功能的特异性突变如何影响其结构和动力学。ttss是许多重要人类病原体的重要毒力决定因素。利用志贺氏菌系统，我们首次描述了III型分泌诱导的不同步骤。这种前所未有的III型分泌步骤的解剖现在为揭示III型分泌诱导的结构基础提供了工具。在这项研究中，我们已经开始合作探索III型分泌水平，目前在任何其他细菌系统中都不可能实现。这里获得的信息将有助于未来设计阻断III型分泌的实用方法。