Assembly/function of the sorting platform of the Shigella type III secretion apparatus

志贺氏菌III型分泌仪分选平台的组装/功能

• 批准号: 9082034
• 负责人: WILLIAM D. PICKING
• 金额: $ 45.44万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-05 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9082034
• 关键词: ATP phosphohydrolase; Age; Anti-Infective Agents; Architecture; Automobile Driving; Bacillary Dysentery; Binding Proteins; Biochemical; Cells; Child; Childhood; Cognitive; Complex; Cytoplasmic Structures; Cytoplasmic Tail; Data; Development; Dysentery; Exhibits; Extracellular Structure; Fluorescence Recovery After Photobleaching; Foundations; Future; Human; Impairment; In Vitro; Infection; Investigation; Knock-out; Large Intestine; Link; Mechanics; Modeling; Molecular; Molecular Chaperones; Morbidity - disease rate; Mutation; Nature; Needles; Peptides; Positioning Attribute; Property; Proteins; Public Health; Recombinants; Research; Role; Shigella; Shigella Infections; Sorting - Cell Movement; Staging; Structure; Testing; Type III Secretion System Pathway; Virulence; Work; base; electron tomography; inhibitor/antagonist; kinetosome; mortality; mutant; nanomachine; null mutation; pathogen; protein protein interaction; public health relevance; small molecule inhibitor; stoichiometry

 DESCRIPTION (provided by applicant): Shigella species cause shigellosis (bacillary dysentery) with high global morbidity and childhood mortality. Shigella uses a type III secretion system (T3SS) to deliver virulence proteins into host cells to promote pathogen entry as the first step in establishing infection. The T3S apparatus (T3SA) consists of: 1) an external needle and its tip complex for delivering translocator and effector proteins; 2) a basal body that spans the bacterial envelope; and 3) a poorly defined cytoplasmic sorting platform that energizes secretion and controls the temporal delivery of secretion substrates. We recently provided the most detailed structural analysis of the Shigella T3SA sorting platform to date, which has allowed us to generate a model for the placement of the Spa47 ATPase, its interaction with the basal body via Spa13, and its association with the other key soring platform components MxiN and Spa33. From the available data, we now hypothesize that the interactions occurring between these components are required for stabilizing the T3SA and that the dynamic nature of these interactions is pivotal for controlling secretion status. Such interactions should be identifiable through ultrastructural analysis of the T3SA in Shigella minicells and by biochemical/molecular analyses. To test our hypothesis, the specific aims of this investigation are 1) to identify the specific protein-protein interactions and biochemical properties of the sorting platform components to determine their roles in controlling type III secretion, 2) to determine the importance of structural changes within the Shigella T3SA sorting platform during different stages of secretion induction and in mutants, and 3) to test proposed models of Shigella type III secretion that implicate sorting platform interactions in activation of the T3SA. These aims will allow us to demonstrate the physical requirements for Spa33 interaction with the cytoplasmic domain of MxiG and the sorting platform component MxiN. Such interactions are necessary for the assembly and stabilization (and thus function) of the T3SA sorting platform and the way these interactions influence the platforms structure as determined by cryo-electron tomography. In parallel, we will test two potentially complementary models by which the platform contributes mechanistically to type III secretion activation. T3SSs are essential virulence determinants for many important human pathogens, but our understanding of the mechanics of these nanomachines remains poor. We will use the Shigella T3SS as a model to uncover the link between secretion status and the role of the T3SA cytoplasmic complex in driving secretion. The interdisciplinary team assembled to complete this work will reveal the fundamental mechanisms by which type III secretion occurs and identify steps that might serve as future targets for anti-infective drugs.

 描述（由申请方提供）：志贺菌属引起志贺菌病（细菌性痢疾），全球发病率和儿童死亡率较高。志贺氏菌使用III型分泌系统（T3 SS）将毒力蛋白递送到宿主细胞中以促进病原体进入作为建立感染的第一步。T3 S装置（T3 SA）由以下组成：1）用于递送转运蛋白和效应蛋白的外部针及其尖端复合物; 2）跨越细菌包膜的基体;和3）不明确的细胞质分选平台，其激发分泌并控制分泌底物的时间递送。我们最近提供了迄今为止志贺氏菌T3 SA分选平台的最详细的结构分析，这使我们能够生成Spa 47 ATP酶的放置模型，其通过Spa 13与基体相互作用，以及其与其他关键分选平台组件MxiN和Spa 33的关联。根据现有数据，我们现在假设这些组分之间发生的相互作用是稳定T3 SA所需的，并且这些相互作用的动态性质对于控制分泌状态至关重要。这种相互作用应通过志贺氏菌小细胞中T3 SA的超微结构分析和生物化学/分子分析来鉴定。为了验证我们的假设，本研究的具体目的是1）鉴定分选平台组分的特异性蛋白质-蛋白质相互作用和生物化学性质，以确定它们在控制III型分泌中的作用，2）确定在分泌诱导的不同阶段和突变体中志贺氏菌T3 SA分选平台内结构变化的重要性，和3）测试所提出的III型志贺氏菌分泌的模型，该模型涉及T3 SA活化中的分选平台相互作用。这些目标将使我们能够证明Spa 33与MxiG的胞质结构域和分选平台组分MxiN相互作用的物理要求。这种相互作用对于T3 SA分选平台的组装和稳定化（以及因此的功能）以及这些相互作用影响平台结构的方式是必需的，如通过冷冻电子断层扫描所确定的。同时，我们将测试两个潜在的互补模型，通过该模型，该平台在机制上有助于III型分泌激活。T3 SSs是许多重要人类病原体的重要毒力决定因素，但我们对这些纳米机器的机制的理解仍然很差。我们将使用志贺氏菌T3 SS作为模型，以揭示分泌状态和T3 SA胞质复合物在驱动分泌中的作用之间的联系。为完成这项工作而组建的跨学科团队将揭示III型分泌发生的基本机制，并确定可能作为抗感染药物未来靶点的步骤。