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（由应用提供）：志贺氏菌物种引起志氏菌病（细菌痢疾），具有较高的全球发病率和儿童死亡率。 Shigella使用III型分泌系统（T3SS）将病毒蛋白输送到宿主细胞中，以促进病原体进入，作为建立感染的第一步。 T3S设备（T3SA）由：1）外针及其尖端复合物用于输送转运剂和效应蛋白； 2）跨细菌包膜的基体； 3）一个定义较差的细胞质分类平台，能够为分泌提供能量并控制分泌底物的临时递送。最近，我们提供了迄今为止志贺氏菌T3SA排序平台的最详细的结构分析，这使我们能够生成一个模型，用于放置SPA47 ATPase，通过SPA13与基本体的相互作用，以及与其他关键刺激平台MXIN和SPA33的关联。从可用的数据中，我们现在假设这些组件之间发生的相互作用是稳定T3SA所必需的，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对志贺氏菌微型赛中T3SA的超微结构分析以及生化/分子分析来确定。为了检验我们的假设，这项研究的具体目的是1）确定排序平台组件的特定蛋白质 - 蛋白质相互作用和生化特性，以确定它们在控制III型分泌中的作用，2）确定结构性变化的重要性，以确定Shigella T3SA分类平台在shigella themiation of Shotirion type sh of Sheriation of Shotion thope of Shotion和3）中的重要性。 T3SA激活中的平台相互作用。这些目标将使我们能够证明与MXIG的细胞质结构域和分类平台组件MXIN相互作用的物理要求。这种相互作用对于T3SA排序平台的组装和稳定（并因此功能）是必要的，并且这些相互作用影响平台结构由低温电子断层扫描确定。同时，我们将测试两个潜在的完整模型，通过该模型，该平台可以通过机械贡献III型分泌激活。 T3SS是许多重要人类病原体的必不可少的毒力确定剂，但是我们对这些纳米机制机制的理解仍然很差。我们将使用Shigella T3SS作为模型来揭示分泌状态与T3SA细胞质复合物在驱动分泌中的作用之间的联系。跨学科的团队组装完成这项工作，将揭示III型分泌发生的基本机制，并确定可能是抗感染药物的未来目标的步骤。