A Mechanism for Shigella Type III Secretion Activation

描述（由申请人提供）：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）将蛋白质递送到宿主细胞中以促进细菌进入。从其在TTSS针尖的位置，侵袭质粒抗原D（iPad）用于控制志贺氏菌III型分泌。通过孵育志贺氏菌与脱氧胆酸盐（DOC），我们已经表明，第一个分泌的转运蛋白，IpaB，可以招募到针尖复合物没有进一步诱导III型分泌。在将细菌与富含胆固醇和鞘磷脂的脂质体一起孵育后，第二转运蛋白（IpaC）被募集到针尖复合物中，这也导致完全诱导III型分泌。因此，志贺氏菌TTSS提供了一种新的模型系统，探索III型分泌诱导的各个步骤。基于这里提出的初步研究结果，我们假设iPad感知环境信号，触发IpaB到针尖复合体的受控募集。由于DOC模拟了III型分泌诱导途径上这一独特步骤所需的环境信号，我们计划确定负责IpaB募集至TTSA针尖复合物的物理和分子机制。为此，本研究的具体目标是：1）解析结合DOC的iPad的晶体结构，以确定负责IpaB募集到志贺氏菌表面的构象变化; 2）靶向iPad上的关键位点，用于突变分析和表型表征;和3）确定靶向突变对iPad的溶液性质的影响，并产生新的晶体和共-iPad 1 DOC晶体，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是许多重要人类病原体的重要毒力决定因子。使用志贺氏菌系统，我们是第一个描述III型分泌诱导的不同步骤。III型分泌步骤的这一前所未有的解剖现在为揭示III型分泌诱导的结构基础提供了工具。在这项研究中，我们已经开始了一项合作努力，以探索III型分泌的水平目前不可能在任何其他细菌系统。在这里获得的信息将有助于未来设计的实用方法阻断III型分泌。 公共卫生相关性：福氏志贺菌是细菌性痢疾（志贺菌病）的病原体，是全球性的公共卫生问题。为了引发感染，S.弗氏使用复杂的III型分泌系统将蛋白质递送到人肠上皮细胞中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，以便有可能确定可以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.