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.

项目摘要 志贺氏菌引起细菌痢疾，具有高世界的发病率和儿童死亡率，并发症 这包括每年患有多次腹泻发作的儿童的认知和发育障碍。 Shigella的主要病毒因素是其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的进入。 T3SS与许多革兰氏阴性病原体共享 符合注入剂的A（n）：1）外部针和尖端复合物，用于传递转运剂和效果； 2） 跨细菌包膜的基本身体； 3）能量和 控制分泌。我们通过冷冻电子层析成像（Cryo-et）启用了SP，从那以后我们 其他人已经确定了sp的组成部分。我们现在建议研究探索结构差异 在原位注射体的“ ON”和“ OFF”分泌状态之间，并平行生化分析 志贺氏菌注射体宿主膜界面的第一个可视化和第一次可视化。我们将 使用Cryo-ET方法以1 nm或更好的分辨率查看SP POD，然后使用生化和分子 当我们开发组装和功能模型时的方法。在我们对SP的投资中，我们将探索 蛋白质结构域在SP界面与内膜环的运动。同时，我们将扩展我们的 研究注射体的另一个重要的细胞质成分 - 形成的出口门非仪表 由MXIA在没有SP的情况下进行结构重排。我们还将利用系统 我们生成了用于捕获原位注射体内不同分泌基材的生成，以便我们可以确定 如何比较三个不同状态的整体结构。这些是“ ON”注射体（iPad null应变） 和两种形式的“ OFF”注射体（MXIH无效应变和效应菌株），它们将提供功能性 深入了解SP和出口门通信和关联。我们提出了三个完整的目标：1） 定义SP/Innermbrane环（IR）界面的化妆，中间状态和结构要求 这允许SP组装和指导III型分泌。 2）将出口门结构特征与分泌相关联 使用完整的冷冻-ET和分子方法的状态； 3）确定相关的结构变化 在原位注射室内捕获底物，并开始生成第一张高分辨率图片 使用冷冻-ET原位注射宿主膜接口。改进的Cryo-ET方法提供了 志贺氏菌注射体内的实体观点原位揭示了不可能的元素 使用纯化的针络合物进行了研究，这反映在此处介绍的初步数据中。我们可以 现在可视化这些子结构，将它们靶向分子分析并净化它们以进行体外生化 和生物物理分析。 T3SS是许多病原体的必需病毒决定因素，但我们仍然缺乏 确定III型分泌基础的机制所需的结构理解。