Pathogen-host Interactions that Remodel the Cytoskeleton

致命的肠道病原体福氏志贺菌篡夺肌动蛋白细胞骨架的成分进入， 在宿主细胞中的运动，并用于形成用于感染邻近细胞的突起。志贺氏菌 蛋白IpaA在细胞接触时从细菌分泌，并且对于细菌摄取是必需的， 发病机制IpaA通过与黏着斑蛋白（一种高度保守的细胞骨架蛋白）结合来完成这些任务 它在指导肌动蛋白网络的组装中起着重要的作用， 基质）和粘附（细胞-细胞）连接。白蛋白通常协调肌动蛋白细胞骨架的变化 通过与其他关键配偶体结合，例如与粘着斑中的整联蛋白受体结合的talin，或与α- 辅肌动蛋白，其在粘附连接中起关键作用。我们最近的研究表明，talin和α-辅肌动蛋白 通过破坏黏着斑蛋白尾部结构域（Vt）与 它的AMerplasty七螺旋束（Vh 1）结构域，通过引起显着和独特的变化， 通过我们创造的螺旋束转换的过程，这个域的结构。IpaA结合 已显示黏着斑蛋白增加其对F-肌动蛋白和去乙酰化肌动蛋白丝的结合亲和力， 我们的新研究表明，这是通过IpaA的能力，特别是增加一个新的功能， 黏着斑蛋白覆盖肌动蛋白丝的有刺末端。重要的是，我们的初步研究还表明， IpaA破坏了Vh 1-Vt相互作用，建立了志贺氏菌激活黏着斑蛋白的机制， IpaA还破坏黏着斑蛋白与talin和α-辅肌动蛋白的结合。这些研究支持了 IpaA结合引起黏着斑蛋白结构的独特变化，破坏黏着斑蛋白形成的模型 粘附连接和局灶性粘附，破坏粘着斑蛋白以执行志贺氏菌必需的功能 发病机制为了精确地定义IpaA激活黏着斑蛋白的机制，在特定的细胞中进行了实验。 目的#1将定义黏着斑蛋白：lpaA复合物的晶体结构。我们的新研究表明， talin和α-辅肌动蛋白与黏着斑蛋白的Vh 1结构域的结合足以触发独特的构象， 在整个分子中发生变化。因此，我们假设IpaA结合也诱导了独特的 全长黏着斑蛋白的结构变化，为了验证这一概念，具体目标#2中的实验将 确定IpaA对全长黏着斑蛋白构象的影响以及它如何改变其构象， 与其有约束力的合作伙伴。最后，破坏黏着斑蛋白与IpaA的相互作用， 对志贺菌的致病机制有着深远的影响。为了解决这一假设，具体目标的实验 #3将确定破坏黏着斑蛋白-lpaA相互作用对志贺氏菌进入、运动性和耐药性的影响。 传播，并在体内志贺菌致病。拟议的实验应确定 IpaA和黏着斑蛋白的相互作用是指导志贺氏菌发病所必需的，希望这些 研究还将为防治这种致命病原体的新战略奠定基础。

The deadly enteropathogen Shigella flexneri usurps components of the actin cytoskeleton for entry and movement in the host cell, and for the formation of protrusions used to infect neighboring cells. The Shigella protein IpaA is secreted from the bacterium upon cell contact and is essential for bacterial uptake and pathogenesis. IpaA accomplishes these tasks by binding to vinculin, a highly conserved cytoskeletal protein that plays essential roles in directing the assembly of actin networks following the formation of focal (cell- matrix) and adherens (cell-cell) junctions. Vinculin normally orchestrates changes in the actin cytoskeleton by binding to other key partners such as talin, which binds to integrin receptors in focal adhesions, or to a- actinin, which plays key roles in adherens junctions. Our recent studies have shown that talin and a-actinin activate vinculin by disrupting the intramolecular hydrophobic interactions of vinculin's tail domain (Vt) with its AMerminal seven-helical bundle (Vh1) domain, by provoking remarkable and unique changes in the structure of this domain through a process we have coined helical bundle conversion. IpaA binding to vinculin has been shown to increase its binding affinity for F-actin and to depolymerize actin filaments, and our new studies have shown that this occurs through IpaA's ability to specifically augment a new function for vinculin in capping the barbed ends of actin filaments. Importantly, our Preliminary Studies have also shown that IpaA disrupts the Vh1-Vt interaction, establishing the mechanism by which Shigella activates vinculin, and that IpaA also disrupts the association of vinculin with talin and a-actinin. These studies support a model whereby IpaA binding provokes unique changes in the structure of vinculin that disrupt the formation of adherens junctions and focal adhesions, subverting vinculin to carry out functions essential for Shigella pathogenesis. To precisely define the mechanism of activation of vinculin by IpaA, experiments in Specific Aim #1 will define the crystal structure of the vinculin:lpaA complex. Our new studies have shown that the binding of talin and a-actinin to vinculin's Vh1 domain is sufficient to trigger unique conformational changes throughout the whole molecule. We therefore hypothesize that IpaA binding also induces unique structural changes in full-length vinculin, and to test this notion the experiments in Specific Aim #2 will determine the effects of IpaA on the conformation of full-length vinculin and how it alters its associations with its binding partners. Finally, disrupting the interactions of vinculin with IpaA should have profound effects on Shigella pathogenesis. To address this hypothesis, experiments in Specific Aim #3 will determine the effects of disrupting the vinculin-lpaA interaction on Shigella entry, motility and spread, and upon Shigella pathogenesis in vivo. The proposed experiments should define the interactions of IpaA and vinculin that are required to direct Shigella pathogenesis, and it is hoped that these studies will also provide the foundation for new strategies to combat this deadly pathogen.