The multiple states of IpaB Shigella type III secretion

描述(申请人提供)：细菌性痢疾的病原体福氏志贺氏菌使用III型分泌系统(T3SS)将毒力蛋白输送到宿主细胞中 促进病原体进入这些细胞。III型分泌器(T3SA)由跨越细菌膜的基底和为转位蛋白和效应蛋白的单向输送提供管道的外部针组成。从它在 T3SA针尖，iPad控制III型分泌的关键第一步--将拳头转运蛋白IPAB重新聚集到针尖。从这里，IPAB与宿主细胞膜成分相互作用，将最终的转位蛋白IPAC动员到针尖，导致完全分泌诱导的开始。志贺氏菌为探索T3SA针尖复合体组装的不同步骤以及控制这一过程的环境因素提供了独特的系统。我们已经解决了志贺氏菌针头、针尖蛋白和IPAB的很大一部分的结构。我们还广泛地研究了这些蛋白质的生化性质，并使用电子显微镜(EM)方法重建了新生的T3SA针尖复合体。在这项调查中，我们将在这个坚实的基础上，在我们对IPAB的了解方面向前迈出重要的一步，应用程序来理解一般的T3SS。我们假设IPAB在T3SA针尖具有不同的结构背景，在那里它采用与iPad相关的低聚状态，这对于其作为志贺氏菌T3SA针尖复合体的功能(例如膜穿透)是必不可少的。为了验证这一假说，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启动的T3SA针尖的原位结构特征；2)确定IPAB在单体状态下的晶体结构以及其一般的低聚结构；3)确定IPAB齐聚在脂质相互作用功能中的作用，并确定参与蛋白质-蛋白质相互作用的区域，这些区域参与了TIP复合体的成熟。当IPAB与细胞质中的伴侣(IpgC)结合时，形成一个稳定的结构，该结构使其排队等待分泌。当募集到T3SA针尖时，它与自己和iPad相互作用，承担宿主细胞接触的传感器的角色。这些受调控的中间状态在可比步骤上类似于其他系统中的中间状态，因此它们代表了III型分泌系统的基本共同机制特征。我们知道IPAB是连接T3SA组装和功能的中心组件，我们的团队正在应用一套集成的不同方法来帮助理解IPAB在III型分泌开始中的复杂作用。这项研究有望揭示T3SS的机制方面可能是抗感染药物的靶点。

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.