Pathogen-host Interactions that Remodel the Cytoskeleton

致命的肠道病原体福氏志贺氏菌侵占肌动蛋白细胞骨架的组件进入并 在宿主细胞中运动，并形成用于感染邻近细胞的突起。志贺氏菌 IPAA蛋白是细菌与细胞接触时分泌的，对细菌的吸收和 发病机制。IPAA通过与纽蛋白结合来完成这些任务，纽蛋白是一种高度保守的细胞骨架蛋白 这在指导肌动蛋白网络在焦点(细胞-细胞)形成后的组装中起着至关重要的作用 基质)和粘连(细胞-细胞)连接。纽蛋白通常协调肌动蛋白细胞骨架的变化。 通过与其他关键伙伴结合，如Talin，它与局灶性粘连中的整合素受体结合，或与- 在粘连连接中起关键作用的肌动蛋白。我们最近的研究表明，Talin和α-Actinin 通过破坏纽蛋白尾区(Vt)的分子内疏水相互作用来激活纽蛋白 它的末端七螺旋束(VH1)结构域，通过引起显著和独特的变化 通过这个结构域的一个过程，我们创造了螺旋丛的转换。IPAA绑定到 纽蛋白已被证明可以增加其与F-肌动蛋白的结合亲和力，并解聚肌动蛋白细丝，以及 我们的新研究表明，这是通过IPAA专门增加了一项新的功能来实现的 肌动蛋白细丝带刺末端的纽蛋白。重要的是，我们的初步研究也表明 IPAA破坏了VH1-VT的相互作用，建立了志贺菌激活纽蛋白的机制， 而且，IPAA还破坏了纽蛋白与Talin和α-Actinin的联系。这些研究支持 IPAA结合引起纽蛋白结构的独特变化从而破坏其形成的模型 粘附性连接和焦点粘连，颠覆纽蛋白执行志贺氏菌必不可少的功能 发病机制。为了准确确定IPAA激活纽蛋白的机制，在特定的实验中 目的#1将定义纽蛋白的晶体结构：lpaA复合体。我们的新研究表明 Talin和α-Actinin与vinculin的VH1结构域的结合足以引发独特的构象 整个分子都会发生变化。因此，我们假设IPAA结合也导致唯一的 全长纽蛋白的结构变化，为了测试这一概念，在特定目标#2中的实验将 确定IPAA对全长纽蛋白构象的影响及其如何改变其 与其具有约束力的伙伴之间的联系。最后，破坏纽蛋白与IPAA的相互作用应该 对志贺氏菌的致病有深远的影响。为了解决这一假设，在特定目标下进行实验 #3将确定破坏vinculin-lpaA相互作用对志贺氏菌进入、动力和 传播，并对志贺氏菌的体内致病机理进行了研究。拟议的实验应该定义 IPAA和纽蛋白的相互作用是指导志贺氏菌致病所必需的，希望这些 研究还将为抗击这种致命病原体的新战略提供基础。

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.