The multiple states of IpaB Shigella type III secretion

IpaB III型志贺菌分泌的多种状态

• 批准号: 8774878
• 负责人: WILLIAM D. PICKING
• 金额: $ 45.75万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-04 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8774878
• 关键词: Adopted; Anti-Infective Agents; Antigens; Automobile Driving; Bacillary Dysentery; Binding; Biochemical; Cell membrane; Cells; Complex; Cytoplasm; Data Set; Disease; Electron Microscopy; Environmental Risk Factor; Event; Foundations; Health; Human; Image; In Situ; In Vitro; Incubated; Individual; Infection; Intestines; Invaded; Investigation; Link; Lipids; Membrane; Methodology; Methods; Molecular; Molecular Chaperones; Needles; Penetration; Plasmids; Positioning Attribute; Process; Property; Proteins; Public Health; Recruitment Activity; Role; Shigella; Shigella flexneri; Structure; System; Testing; Type III Secretion System Pathway; Virulence; base; bile salts; in vivo; kinetosome; pathogen; prevent; protein protein interaction; reconstruction; sensor

DESCRIPTION (provided by applicant): Shigella flexneri, the causative agent of bacillary dysentery, uses a type III secretion system (T3SS) to deliver virulence proteins into host cells to promote pathogen entry into these cells. The type III secretion apparatus (T3SA) consists of a basal body that spans both bacterial membranes and an external needle that provides the conduit for unidirectional delivery of translocator and effector proteins. From its position at the T3SA needle tip, IpaD controls the critical first step of type III secretion -recruitment of the fist translocator protein, IpaB, to the needle tip. From here, IpaB interacts with host cell membrane components to mobilize the final translocator protein, IpaC, to the needle tip, resulting in the onset of full secretion induction. Shigella provides a unique system for exploring the distinct steps of T3SA needle tip complex assembly and the environmental factors that control this process. We have solved the structures of the Shigella needle, needle tip proteins and a significant portion of IpaB. We have also extensively examined the biochemical properties of these proteins and used electron microscopy (EM) methods to reconstruct the nascent T3SA needle tip complex. In this investigation, we will build upon this substantial foundation to provid a significant step forward in what we know about IpaB with application for understanding T3SSs in general. We hypothesize that IpaB assumes a distinct structural context at the T3SA needle tip where it adopts an IpaD-associated oligomeric state that is essential for its function (e.g. membrane penetration) as part of the Shigella T3SA needle tip complex. To test this hypothesis, the specific aims of this investigation are: 1) Determine the in situ structural features of the primed T3SA needle tip using electron microscopy-based reconstruction; 2) Determine the crystal structure of IpaB in its monomeric state along with its general oligomeric structure; 3) Determine the role of IpaB oligomerization in lipid-interaction functions and determine regions involved in the protein-protein interactions involved in tip complex maturation. IpaB forms a stable structure when bound by its chaperone (IpgC) in the cytoplasm and this structure queues it for secretion. Upon recruitment to the T3SA needle tip, it interacts with itself and IpaD to assume the role of sensor of host cell contact. These regulated intermediary states would be similar to those in other systems at comparable steps and they thus represent a fundamental shared mechanistic feature of type III secretion systems. We know that IpaB is the central component that links T3SA assembly and function and our team is applying an integrated set of diverse methodologies to help understand the complex role of IpaB in the onset of type III secretion. This study is anticipated to reveal mechanistic aspects of T3SS may be targeted by anti-infective agents.

描述（由申请人提供）：福氏志贺菌是细菌性痢疾的病原体，使用III型分泌系统（T3 SS）将毒力蛋白递送到宿主细胞中， 促进病原体进入这些细胞。III型分泌器（T3 SA）由跨越细菌膜的基体和提供用于单向递送转运蛋白和效应蛋白的导管的外部针组成。从其在 T3 SA针尖，iPad控制III型分泌的关键第一步-将第一个转运蛋白IpaB募集到针尖。从这里开始，IpaB与宿主细胞膜组分相互作用，将最终的转运蛋白IpaC动员到针尖，导致完全分泌诱导的开始。志贺氏菌提供了一个独特的系统，用于探索T3 SA针尖复合物组装的不同步骤和控制该过程的环境因素。我们已经解决了志贺氏菌针，针尖蛋白和IpaB的重要部分的结构。我们还广泛地研究了这些蛋白质的生化特性，并使用电子显微镜（EM）的方法来重建新生的T3 SA针尖复合物。在这项调查中，我们将建立在这个坚实的基础上，提供了一个重要的一步，我们知道IpaB与应用程序的理解T3 SS一般。我们假设IpaB在T3 SA针尖处呈现不同的结构背景，其中它采用iPad相关的寡聚状态，这对其作为志贺氏菌T3 SA针尖复合物的一部分的功能（例如膜穿透）是必不可少的。为了验证这一假设，本研究的具体目的是：1）使用基于电子显微镜的重建来确定引发的T3 SA针尖的原位结构特征; 2）确定IpaB在其单体状态下的晶体结构以及其一般低聚物结构沿着; 3）确定IpaB寡聚化在脂质相互作用功能中的作用，并确定参与蛋白质相互作用的区域。蛋白质相互作用参与尖端复合物成熟。IpaB在细胞质中被其伴侣蛋白（IpgC）结合时形成稳定的结构，并且该结构使其排队以便分泌。在募集到T3 SA针尖时，其与自身和iPad相互作用以承担宿主细胞接触的传感器的作用。这些受调节的中间状态与其他系统中的类似步骤相似，因此它们代表了III型分泌系统的基本共享机制特征。我们知道IpaB是连接T3 SA组装和功能的核心成分，我们的团队正在应用一套综合的不同方法来帮助理解IpaB在III型分泌开始时的复杂作用。这项研究预计将揭示T3 SS的机制方面可能是抗感染药物的目标。