LISTERIA USES HOST CELL ACTIN TO SPREAD CELL TO CELL

李斯特菌利用宿主细胞肌动蛋白在细胞之间传播

• 批准号: 2672226
• 负责人: Frederick s Southwick
• 金额: $ 23.17万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672226
• 关键词: Listeria; Listeria infections; Xenopus oocyte; actin binding protein; actins; bacterial cytopathogenic effect; bacterial proteins; cell motility; cytoskeleton; gene mutation; host organism interaction; microinjections; nucleic acid sequence; phosphoproteins; protein purification; protein structure function; site directed mutagenesis; time resolved data; transfection; video microscopy; vinculin

Listeria and Shigella hijack the actin cytoskeleton of their host cell and use it to spread from cell to cell. Act A, a Listeria surface protein is the only bacterial protein required for assembly of the actin motor and it interacts directly with VASP, a host cytoskeletal protein. This interaction requires at least one of the 4 tandem proline repeat motifs found in ActA. Profilin, an actin binding protein that has several potent effects on actin assembly, binds directly to VASP. This interaction is mediated by different tandem proline repeat motifs found on VASP, and can be blocked by these proline motifs in in vivo assays. Act A, profilin, and VASP accumulate at the interface between the bacterial surface and the growing actin tail. This proposal aims to identify mechanisms by which these protein:protein interactions enable Listeria and Shigella to assemble actin-based motors at their surfaces. The investigator proposes to investigate the nature of these interactions between ActA, VASP, and profilin through a series of genetic alterations of the VASP binding domain on ActA combined with cell biological and biochemical assays. this is a research area that will deepen understanding not only of particular pathogenic processes, but also of the molecular basis for regulation of the actin cytoskeleton to achieve cell motility. There are three specific aims. Aim 1 is to study the binding of ActA, the bacterial surface protein which is the only bacterial protein required for bacterial motility to a host cell protein VASP (vasodilator-stimulated phosphoprotein), already known to bind to ActA and also necessary for motility. Listeria must then, attract VASP to ActA. ActA has four oligoproline sequences separated by stretches of amino acids, (the first, DFPPPPTDE and the other 3 are similar, one has only 3 P interrupted with I); thus prolines with an aromatic group at the start and flanked by negatively charged residues. This is the critical VASP binding site and all 4 are thought to operate but this is not proven. Using PCR site-direct mutagenesis and bacterial expression, the PI proposes to study binding by gel filtration and analytical centrifugation to identify the primary structural determinants and binding constants for the complex between ActA and VASP. Oligoproline analogues will be prepared and studied as competitive inhibitors of complex formation. Aim 2 combines two aims: the binding of vinculin to VASP and ActA function in vivo. The thought is that Listeria must attract VASP from the vinculin binding in the host. Therefore in vitro binding studies will determine the relative affinity of VASP to vinculin vs. ActA. The rank order of peptide analogue inhibition of ActA-VASP and Vinculin-ActA binding will also be studied in vivo by microinjection and time-lapse video microscopy. Listeria ActA mutants will be prepared with a shuttle vector PMK4 and homologous recombinations made with defined changes in the oligoproline repeat sequences. The ability of each mutant bacterium to generate actin tails, move within and spread from cell to cell and to cause disease in mice will be studied. Aim 3 is to study the binding of VASP to profilin and the interaction of this complex with ActA. The idea is that profilin binding to VASP or VASP-ActA may alter the ability of profilin to enhance ATP/ADP exchange on actin monomers.

李斯特菌和志贺氏菌劫持宿主的肌动蛋白细胞骨架 细胞并用它从一个细胞传播到另一个细胞。 A 幕，李斯特菌 表面蛋白是细菌所需的唯一蛋白质 肌动蛋白马达的组装，它直接与 VASP 相互作用， 宿主细胞骨架蛋白。 这种交互至少需要 ActA 中发现的 4 个串联脯氨酸重复基序之一。 Profilin，一种肌动蛋白结合蛋白，具有多种有效作用 对肌动蛋白组装的影响，直接与 VASP 结合。 这 相互作用是由不同的串联脯氨酸重复基序介导的 在 VASP 上发现，并且可以被这些脯氨酸基序阻断 体内测定。 Act A、profilin 和 VASP 在 细菌表面和生长的肌动蛋白之间的界面 尾巴。 该提案旨在确定这些机制 蛋白质：蛋白质相互作用使李斯特菌和志贺氏菌能够 在其表面组装基于肌动蛋白的马达。 调查员 提议调查这些相互作用的性质 ActA、VASP 和 profilin 通过一系列基因改变 ActA 上的 VASP 结合域结合细胞生物学 和生化测定。 这是一个将深化的研究领域 不仅了解特定的致病过程，而且 还研究了肌动蛋白调节的分子基础 细胞骨架实现细胞运动。 具体目标有三个。目标1是研究结合 ActA，细菌表面蛋白，是唯一的细菌 细菌向宿主细胞运动所需的蛋白质 VASP（血管舒张刺激磷蛋白），已知 与 ActA 结合，也是运动所必需的。 李斯特菌必须 然后，将VASP吸引到ActA。 ActA 有四个寡脯氨酸序列 由氨基酸序列分开（第一个，DFPPPPPTDE 和 其他3个类似，其中一个只有3个P，用I打断）； 因此，脯氨酸在起始处具有芳香基团，两侧是 带负电的残基。 这是关键的 VASP 绑定 站点和所有 4 个站点均被认为可以运行，但这尚未得到证实。 利用 PCR 定点诱变和细菌表达， PI 建议通过凝胶过滤和分析来研究结合 离心以确定主要结构决定因素 以及 ActA 和 VASP 之间复合物的结合常数。 寡脯氨酸类似物的制备和研究如下 复合物形成的竞争性抑制剂。 目标 2 结合了两个目标：纽蛋白与 VASP 和 ActA 的结合 在体内发挥作用。 我们的想法是李斯特菌必须吸引 VASP 来自宿主中结合的纽蛋白。 因此在体外 结合研究将确定 VASP 的相对亲和力 纽蛋白与 ActA。 肽类似物抑制的排序 ActA-VASP 和 Vinculin-ActA 结合的研究也将在 通过显微注射和延时视频显微镜进行体内观察。 李斯特菌 ActA 突变体将用穿梭载体 PMK4 和 通过定义的变化进行同源重组 寡脯氨酸重复序列。 每个突变体的能力 细菌产生肌动蛋白尾部，在内部移动并从其传播 将研究细胞与细胞之间以及引起小鼠疾病的情况。 目标 3 是研究 VASP 与 profilin 的结合以及 该复合物与 ActA 的相互作用。 这个想法是 与 VASP 或 VASP-ActA 结合可能会改变 profilin 的能力 增强肌动蛋白单体上的 ATP/ADP 交换。