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: 10673048
• 负责人: Jun Liu
• 金额: $ 61.68万
• 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/10673048
• 关键词: Adaptor Signaling Protein; Age; Amino Acids; Architecture; Biochemical; Cell membrane; Cells; Child; Childhood; Cognitive; Communication; Complement; Complex; Cryo-electron tomography; Cytoplasm; 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; Structure; Structure-Activity Relationship; System; Tertiary Protein Structure; Testing; Type III Secretion System Pathway; Virulence; Virulence Factors; Visualization; biophysical analysis; biophysical techniques; 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.

项目总结 志贺氏菌引起细菌性痢疾，在全球范围内发病率和儿童死亡率高，并伴有并发症 这包括每年遭受多次腹泻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(T3SS)，该系统用于运送效应蛋白 进入宿主细胞以促进病原体进入。许多革兰氏阴性病原体都有T3SS， 注射剂，包括(N)：1)外针和针尖复合体，用于输送移位器和效应器；2) 和3)细胞质分选平台(SP)，它为细菌被膜提供能量并 控制分泌。我们率先通过冷冻电子断层扫描(CRYO-ET)对SP进行可视化，从那时起我们 其他人已经确定了SP的组件。我们现在提出研究，以探索结构性差异。 SP平行生化分析在原位注射剂“开”和“关”分泌状态之间的作用 亚组装和首次在原位可视化志贺氏菌注射体-宿主膜界面。我们会 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 方法当我们开发装配和功能模型时。在我们对SP的调查中，我们将探索 SP与内膜环交界处蛋白质结构域的移动。同时，我们将延长我们的 检测注射体的另一个重要细胞质成分--出口门九聚体的研究 MxiA，在SP缺席的情况下进行结构重组。我们还将利用一个系统 我们已经生成了在原位注射器中捕获不同分泌底物的方法，以便我们可以确定 三个不同州的整体结构如何比较。这些都是“On”注射剂(iPad Null品系) 和两种形式的注射小体(mxiH无效菌株和效应器阻断菌株)，这将提供功能 深入了解SP和出口关口的通信和关联。我们提出了三个互补的目标：1) 定义SP/内膜环(IR)界面的组成、中间状态和结构要求 允许SP组装和引导III型分泌。2)出口门结构特征与分泌物的关系 使用补充冷冻-ET和分子方法的状态；以及3)确定相关的结构变化 在原位注射剂内捕获底物，并开始生成第一张高分辨率图像 用冷冻电子显微镜原位观察注射体-宿主膜界面。改进的冷冻-ET方法提供了一种 史无前例地原位观察志贺氏菌注射体内的亚结构，以揭示不能 使用提纯的针状化合物进行了研究，这反映在这里提供的初步数据中。我们可以的 现在将这些亚结构可视化，针对它们进行分子分析，并纯化它们以进行体外生化 和生物物理分析。T3SS是许多病原体必不可少的毒力决定因素，但我们仍然缺乏 对结构的理解需要确定III型分泌物的基础机制。