A Mechanism for Shigella Type III Secretion Activation

III 型志贺氏菌分泌激活机制

基本信息

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
项目类别：
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

项目摘要

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）：芽孢杆菌痢疾的病因志贺氏菌Flexneri使用其III型分泌系统（TTSS）将蛋白质输送到宿主细胞中以促进细菌进入。从其在TTSS针尖的位置，入侵质粒抗原D（iPad）可以控制III型志贺氏菌分泌。通过将志贺氏菌与脱氧胆酸（DOC）孵育，我们表明，可以将第一个分泌的转运蛋白IPAB募集到针尖复合体中，而无需进一步诱导III型分泌。将第二个转运剂（IPAC）募集到针尖复合物中是在将细菌与富含胆固醇和鞘磷脂的脂质体孵育后，这也导致III型分泌的完全诱导。因此，Shigella TTSS提供了一种新型的模型系统，用于探索III型分泌诱导的单个步骤。根据此处介绍的初步发现，我们假设iPad感知环境信号以触发IPAB受控募集到针尖复合体中。由于DOC模仿了III型分泌诱导途径上这一独特步骤所需的环境信号，因此我们计划确定负责IPAB募集到TTSA针头尖端复合物的物理和分子机制。为此，这项研究的具体目的是：1）与DOC结合使用iPad的晶体结构，以识别导致IPAB募集到Shigella表面的构象变化； 2）iPad上的关键位点用于突变分析和表型表征； 3）确定靶向突变对iPad iPad的溶液特性的影响，并生成新的晶体和iPad 1 doc的共结晶，以确定改变iPad功能的特定突变如何影响其结构和动态。 TTSS是许多重要人类病原体的必不可少的毒力决定因素。使用志贺氏菌系统，我们是第一个描述III型分泌诱导的不同步骤的人。现在，对III型分泌步骤的前所未有的解剖现在为揭示III型分泌诱导的结构基础提供了一种工具。在这项调查中，我们采取了协作努力，以探索III型分泌的某种水平，目前在任何其他细菌系统中都无法进行。这里获得的信息将有助于未来的实用方法，以阻止III型分泌。公共卫生相关性：Shigella Flexneri是全球公共卫生问题，是杆菌痢疾（Shigellosis）的病因。为了引发感染，S。flexneri使用复杂的III型分泌系统将蛋白质输送到人肠上皮细胞中，以促进细菌进入这些细胞。在这项研究中，我们将确定该分泌系统被激活的机制，以便可以识别可以中和志贺氏菌引起人类疾病能力的化合物。