Characterization of assembly and activation of the Shigella type III secretion injectisome

项目摘要 志贺氏菌引起的细菌性痢疾在世界范围内发病率和儿童死亡率都很高，并伴有并发症 其中包括每年遭受多次癫痫发作的儿童的认知和发育障碍。 志贺氏菌的主要毒力因子是其III型分泌系统（T3 SS），其用于递送效应蛋白 进入宿主细胞以促进病原体进入。T3 SS由许多革兰氏阴性病原体共享， 注射器，其包括：1）用于递送移位器和效应器的外部针和尖端复合体; 2） 跨越细菌包膜的基体;和3）细胞质分选平台（SP），其为细菌的细胞质分选提供能量， 控制分泌。我们率先通过冷冻电子断层扫描（cryo-ET）可视化SP，从那时起， 和其他人已经确定了SP的组成部分。我们现在提出研究，以探索结构差异 在原位注射器的“开”和“关”分泌状态之间，同时进行SP的生化分析 子组件和志贺氏菌注射体-宿主膜界面的首次原位可视化。我们将 使用冷冻ET方法以1 nm或更好的分辨率观察SP吊舱，然后使用生物化学和分子生物学方法， 方法，因为我们开发的组装和功能模型。在我们对SP的调查中，我们将探讨 SP界面处的蛋白质结构域与内膜环的运动。与此同时，我们将扩大我们的 研究检查注射体的另一个重要细胞质组分-形成的输出门九聚体 通过MxiA，它在没有SP的情况下经历结构重排。我们还将利用一个系统 在原位注射器中捕获不同的分泌底物， 三个不同州的整体结构比较。这些是“开启”注射体（iPad无效菌株） 和两种形式的“关闭”注射体（mxiH无效菌株和效应子阻断菌株），其将提供功能性的 深入了解SP和出口门通信和关联。我们提出三个互补的目标：1） 定义SP/内膜环（IR）界面的组成、中间状态和结构要求 其允许SP组装并引导III型分泌。2)将出口门结构特征与分泌相关联 使用互补的cryo-ET和分子方法的状态;和3）确定相关的结构变化 并开始产生第一高分辨率图像 注射体-宿主膜界面原位使用冷冻ET。改进的冷冻ET方法提供了一种 志贺氏菌注射体内的亚结构的前所未有的观点，以揭示不能被 使用纯化的针复合物进行研究，这反映在这里提供的初步数据中。我们可以 现在可视化这些亚结构，将它们作为分子分析的目标，并将它们纯化用于体外生化 和生物物理分析。T3 SS是许多病原体的重要毒力决定因子，但我们仍然缺乏 结构的理解需要确定的机制，根据III型分泌。

PROJECT SUMMARY Shigella causes bacillary dysentery with high worldwide morbidity and childhood mortality with complications that include cognitive and developmental impairment in children suffering multiple diarrheal episodes each year. Shigella’s main virulence factor is its type III secretion system (T3SS), which is used to deliver effector proteins into host cells to promote pathogen entry. T3SSs are shared by many Gram negative pathogens with the injectisome comprising a(n): 1) external needle and tip complex for delivering translocators and effectors; 2) basal body that spans the bacterial envelope; and 3) cytoplasmic sorting platform (SP) that energizes and controls secretion. We pioneered visualizing the SP by cryo-electron tomography (cryo-ET) and since then we and others have identified the components of the SP. We now propose studies to explore structural differences between the “on” and “off” secretion states for the in situ injectisome with parallel biochemical analysis of the SP sub-assemblies and the first visualization of the Shigella injectisome-host membrane interface in situ. We will use cryo-ET methods to view the SP pods at a 1-nm or better resolution and then use biochemical and molecular methods as we develop models of assembly and function. In our investigation of the SP, we will explore the movement of protein domains at the SP interface with the inner membrane ring. In parallel, we will extend our study to examine another important cytoplasmic component of the injectisome - the export gate nonamer formed by MxiA, which undergoes structural rearrangements in the absence of the SP. We will also exploit a system we’ve generated for trapping different secretion substrates within the in situ injectisome so that we can determine how the overall structure compares for three different states. These are the “on” injectisome (ipaD null strain) and two forms of “off” injectisomes (mxiH null strain and effector-blocked strains), which will provide functional insight into SP and export gate communication and association. We propose three complementary aims: 1) Define the makeup, intermediate states and structural requirements at the SP/inner-membrane ring (IR) interface that allow SP assembly and guide type III secretion. 2) Correlate export gate structural features with secretion status using complementary cryo-ET and molecular methods; and 3) Identify the structural changes associated with trapping substrates within the in situ injectisome and begin generating the first high-resolution picture of the injectisome-host membrane interface in situ using cryo-ET. Improved cryo-ET methods provide an unprecedented view of substructures within the Shigella injectisome in situ to reveal elements that cannot be studied using purified needle complexes and this is reflected in the preliminary data presented here. We can now visualize these sub-structures, target them for molecular analysis and purify them for in vitro biochemical and biophysical analysis. The T3SS is an essential virulence determinant for many pathogens, but we still lack the structural understanding needed to determine the mechanisms that underlie type III secretion.