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

III 型志贺氏菌分泌注射剂的组装和激活的表征

• 批准号: 10535257
• 负责人: Jun Liu
• 金额: $ 63.09万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535257
• 关键词: Adaptor Signaling Protein; Age; Amino Acids; Architecture; Biochemical; Cell membrane; Cells; Child; Childhood; Cognitive; Communication; Complement; Complex; Cryo-electron tomography; Data; Development; Diarrhea; Dissection; Dysentery; Elements; Ensure; Event; Foundations; Future; Image; Impairment; In Situ; In Vitro; Incidence; Investigation; Knowledge; Large Intestine; Learning; Membrane; Methods; Modeling; Molecular; Molecular Analysis; Morbidity - disease rate; Movement; Mutation Analysis; Nature; Needles; Positioning Attribute; Process; Proteins; Public Health; Research; Resolution; Shigella; Shigella Infections; Sorting - Cell Movement; Structure; Structure-Activity Relationship; System; Tertiary Protein Structure; Testing; Type III Secretion System Pathway; Virulence; Virulence Factors; Visualization; biophysical analysis; biophysical techniques; density; flexibility; imaging approach; imaging modality; improved; insight; kinetosome; mortality; mutant; nanomachine; new therapeutic target; pathogen; protein protein interaction; small molecule inhibitor

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.

项目概要 志贺氏菌引起细菌性痢疾，全球发病率和儿童死亡率很高，并伴有并发症 其中包括每年多次腹泻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 (T3SS)，用于传递效应蛋白 进入宿主细胞以促进病原体进入。许多革兰氏阴性病原体共有 T3SS 注射体，包含a(n)：1)用于递送易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； 3) 细胞质分选平台 (SP) 控制分泌。我们率先通过冷冻电子断层扫描 (cryo-ET) 可视化 SP，从那时起我们 等人已经确定了 SP 的组成部分。我们现在提出研究来探索结构差异 通过 SP 的平行生化分析，确定原位注射体的“开”和“关”分泌状态 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 或更高的分辨率查看 SP pod，然后使用生化和分子 我们开发装配和功能模型时的方法。在我们对 SP 的调查中，我们将探讨 SP 与内膜环界面处的蛋白质结构域的运动。与此同时，我们将扩展我们的 研究检查注射体的另一个重要的细胞质成分 - 形成的出口门九聚体 由 MxiA 产生，在没有 SP 的情况下会发生结构重排。我们还将利用一个系统 我们已经生成了用于在原位注射体内捕获不同分泌底物的方法，以便我们可以确定 三种不同状态的整体结构如何比较。这些是“on”注射体（ipaD 无效菌株） 以及两种形式的“关闭”注射体（mxiH 无效菌株和效应子阻断菌株），这将提供功能性 深入了解 SP 和出口门的沟通和关联。我们提出三个互补目标：1) 定义 SP/内膜环 (IR) 界面的构成、中间状态和结构要求 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 使用互补的冷冻电子断层扫描和分子方法的状态； 3) 确定相关的结构变化 将底物捕获在原位注射体内，并开始生成第一张高分辨率图像 使用冷冻电子断层扫描（cryo-ET）原位注射体-宿主膜界面。改进的冷冻 ET 方法提供了 对志贺氏菌注射体内的亚结构进行前所未有的观察，以揭示无法识别的元素 使用纯化的针复合物进行了研究，这反映在此处提供的初步数据中。我们可以 现在可视化这些子结构，将它们作为分子分析的目标并纯化它们以用于体外生化 和生物物理分析。 T3SS是许多病原体的重要毒力决定因素，但我们仍然缺乏 确定 III 型分泌机制所需的结构理解。