The multiple states of IpaB Shigella type III secretion

IpaB III型志贺氏菌分泌的多种状态

• 批准号: 8442553
• 负责人: WILLIAM D. PICKING
• 金额: $ 45.47万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 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/8442553
• 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; 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; public health relevance; 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）：细菌痢疾的病因Shigella Flexneri使用III型分泌系统（T3SS）将毒力蛋白传递到宿主细胞中 促进病原体进入这些细胞。 III型分泌设备（T3SA）由一个基础体组成，该体体跨越细菌膜和外针，可为单向递送转运剂和效应蛋白提供管道。从其位置 T3SA针尖，iPad控制III型分泌的关键第一步 - 拳头易位蛋白IPAB的摄取率向针尖。从这里开始，IPAB与宿主细胞膜成分相互作用，以将最终的转运蛋白IPAC动员到针尖，从而导致全部分泌诱导的发作。 Shigella提供了一个独特的系统，用于探索T3SA针尖复合物组件的不同步骤以及控制此过程的环境因素。我们已经解决了志贺氏菌针头，针尖蛋白和大部分IPAB的结构。我们还广泛研究了这些蛋白质的生化特性，并使用了电子显微镜（EM）方法来重建新生的T3SA针头尖端复合物。在这项调查中，我们将基于这一基础，为我们对IPAB的了解迈出了重要的一步，并以理解T3SS的申请为基础。我们假设IPAB在T3SA针尖上采用独特的结构环境，在该尖端中采用了与iPad相关的寡聚状态，这对于其功能至关重要（例如膜穿透）作为Shigella T3SA针尖的一部分。为了检验这一假设，该研究的具体目的是：1）使用基于电子显微镜的重建来确定尖头T3SA针尖的原位结构特征； 2）确定IPAB在其单体状态的晶体结构以及其一般的寡聚结构； 3）确定IPAB寡聚在脂质相互作用函数中的作用，并确定参与尖端复杂成熟涉及的蛋白质 - 蛋白质相互作用的区域。当由细胞质中的伴侣（IPGC）绑定时，IPAB形成稳定的结构，并且该结构排队为分泌。募集到T3SA针尖后，它与自身相互作用，并在宿主细胞接触的传感器中发挥作用。这些受调节的中间状态将与其他系统中的其他系统相似，因此它们代表了III型分泌系统的基本共同机械特征。我们知道IPAB是链接T3SA组装和功能的中心组件，我们的团队正在应用一组集成的方法，以帮助了解IPAB在III型分泌型发作中的复杂作用。预计这项研究将揭示T3SS的机械方面可能是抗感染剂的目标。