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

 描述(申请人提供)：志贺氏菌属引起志贺氏菌病(细菌性痢疾)，具有较高的全球发病率和儿童死亡率。志贺氏菌使用III型分泌系统(T3SS)将毒力蛋白输送到宿主细胞中，以促进病原体进入，这是建立感染的第一步。T3S装置(T3SA)包括：1)外针及其顶端复合体，用于输送转运子和效应器蛋白；2)跨越细菌被膜的基底；以及3)定义不明确的细胞质分选平台，其为分泌提供能量并控制分泌底物的临时输送。我们最近提供了迄今为止最详细的志贺氏菌T3SA分选平台的结构分析，使我们能够生成Spa47 ATPase的放置模型，它通过Spa13与基础体的相互作用，以及它与其他关键分选平台组件MxiN和Spa33的关联。根据现有数据，我们现在假设，这些成分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。这种相互作用应该可以通过对志贺氏菌微细胞中T3SA的超微结构分析和生化/分子分析来识别。为了验证我们的假设，本研究的具体目标是1)确定特定的蛋白质-蛋白质相互作用和分选平台组件的生化性质，以确定它们在控制III型分泌中的作用；2)确定志贺氏菌T3SA分选平台在不同分泌诱导阶段和突变体中结构变化的重要性；以及3)测试所提出的志贺菌III型分泌模型，该模型涉及分选平台相互作用在T3SA激活中的作用。这些目的将使我们能够证明Spa33与MxiG的细胞质结构域和分选平台组件MxiN相互作用的物理要求。这种相互作用对于T3SA分选平台的组装和稳定(从而发挥作用)以及这些相互作用影响冷冻电子断层扫描确定的平台结构的方式是必要的。同时，我们将测试两个潜在的互补模型，通过这两个模型，平台机械地促进III型分泌激活。T3SS是许多重要的人类病原体的基本毒力决定因素，但我们对这些纳米机器的机制了解仍然很差。我们将以志贺氏菌T3SS为模型，揭示分泌状态与T3SA细胞质复合体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型分泌物发生的基本机制，并确定可能作为未来抗感染药物靶点的步骤。

 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.