A Mechanism for Shigella Type III Secretion Activation

描述(申请人提供)：福氏志贺菌是细菌性痢疾的病原体，利用其III型分泌系统(TTSS)将蛋白质输送到宿主细胞中，促进细菌进入。侵袭质粒抗原D(IPad)从其位于TTSS针尖的位置起到控制志贺菌III型分泌的作用。通过将志贺菌与脱氧胆酸盐(DOC)孵育，我们已经证明，第一个分泌的转运蛋白IPAB可以被招募到针尖复合体中，而不会进一步诱导III型分泌。当细菌与富含胆固醇和鞘磷脂的脂质体孵育时，第二转位蛋白(IPAC)被招募到针尖复合体，这也导致III型分泌的完全诱导。因此，志贺氏菌TTSS为探索III型分泌诱导的各个步骤提供了一个新的模型系统。根据这里提出的初步发现，我们假设iPad感知环境信号，从而触发IPAB向针尖复合体的受控招募。由于DOC模拟了诱导III型分泌的这一不同步骤所需的环境信号(S)，因此我们计划确定导致IPAB募集到TTSA针尖复合体的物理和分子机制。为此，本次研究的具体目的是：1)解决与DOC结合的iPad的晶体结构，以确定导致IPAB聚集到志贺氏菌表面的构象变化；2)针对iPad上的关键位点进行突变分析和表型表征；以及3)确定靶向突变对iPad溶液性能的影响，并生成iPad 1 DOC的新晶体和共晶，以确定改变iPad功能的特定突变如何影响其结构和动力学。TTSS是许多重要的人类病原体的重要毒力决定因素。使用志贺氏菌系统，我们第一次描述了III型分泌诱导的不同步骤。这一对III型分泌步骤的史无前例的剖析现在为揭示III型分泌诱导的结构基础提供了一个工具。在这项研究中，我们开始了一项合作努力，以探索III型分泌物的水平，目前任何其他细菌系统都不可能。所获得的信息将有助于未来设计实用的方法来阻断III型分泌。 与公共卫生相关：福氏志贺菌是全球公共卫生问题，是细菌性痢疾(志贺氏菌病)的病原体。为了启动感染，福氏志贺氏菌使用一种复杂的III型分泌系统将蛋白质输送到人类肠道上皮细胞，以促进细菌进入这些细胞。在这项研究中，我们将确定这种分泌系统被激活的机制，以便有可能识别出能够中和志贺氏菌引起人类疾病的能力的化合物。

DESCRIPTION (provided by applicant): Shigella flexneri, the causative agent of bacillary dysentery, uses its type III secretion system (TTSS) to deliver proteins into host cells to promote bacterial entry. From its position at the tip of the TTSS needle, invasion plasmid antigen D (IpaD) serves to control Shigella type III secretion. By incubating Shigella with deoxycholate (DOC), we have shown that the first secreted translocator protein, IpaB, can be recruited to the needle tip complex without further induction of type III secretion. Recruitment of the second translocator (IpaC) to the needle tip complex occurs upon incubating the bacteria with liposomes rich in cholesterol and sphingomyelin, which also results in full induction of type III secretion. Thus, the Shigella TTSS provides a novel model system for exploring the individual steps of type III secretion induction. Based on the preliminary findings presented here, we hypothesize that IpaD senses environmental signals to trigger the controlled recruitment of IpaB to the needle tip complex. Because DOC mimics the environmental signal(s) needed for this distinct step on the pathway to type III secretion induction, we plan to determine the physical and molecular mechanism responsible for IpaB recruitment to the TTSA needle tip complex. To do this, the specific aims of this investigation are to: 1) solve the crystal structure of IpaD with DOC bound to identify the conformational changes responsible for IpaB recruitment to the Shigella surface; 2) target key sites on IpaD for mutational analysis and phenotypic characterization; and 3) determine the influence of targeted mutations on solution properties of IpaD IpaD and generate new crystals and co-crystals of IpaD 1 DOC to determine how specific mutations that alter IpaD function affect its structure and dynamics. TTSSs are essential virulence determinants for many significant human pathogens. Using the Shigella system, we are the first to describe the distinct steps of type III secretion induction. This unprecedented dissection of the steps of type III secretion now provides a tool for revealing the structural basis for type III secretion induction. In this investigation, we have embarked on a collaborative effort to explore type III secretion at a level not currently possible in any other bacterial system. The information obtained here will contribute to the future design of practical methods for blocking type III secretion. PUBLIC HEALTH RELEVANCE: Shigella flexneri is global public health problem as the causative agent of bacillary dysentery (shigellosis). To initiate infection, S. flexneri uses a complex type III secretion system to deliver proteins into human intestinal epithelial cells to promote bacterial entry into these cells. In this investigation, we will identify the mechanism by which this secretion system becomes activated so that it may be possible to identify compounds that can neutralize Shigella's ability to cause human disease.