LISTERIA USES HOST CELL ACTIN TO SPREAD CELL TO CELL

李斯特菌和志贺氏菌劫持宿主的肌动蛋白细胞骨架 并利用它在细胞间传播。 A幕，李斯特菌 表面蛋白是唯一的细菌蛋白质所需的 肌动蛋白马达的组装，它直接与VASP相互作用， 一种宿主细胞骨架蛋白。 这种互动至少需要 ActA中发现的4个串联脯氨酸重复基序之一。 Profilin是一种肌动蛋白结合蛋白，具有几种有效的作用 对肌动蛋白装配的影响，直接与VASP结合。 这 相互作用由不同的串联脯氨酸重复基序介导 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 本建议旨在确定这些机制， 蛋白质：蛋白质相互作用使李斯特菌和志贺氏菌能够 在表面组装肌动蛋白发动机。 研究者 建议调查这些相互作用的性质， ActA、VASP和profilin通过一系列遗传改变 ActA上的VASP结合结构域与细胞生物学特性结合的方法 和生化分析。 这是一个研究领域， 不仅了解特定的致病过程， 也是调节肌动蛋白的分子基础 细胞骨架以实现细胞运动。 有三个具体目标。目的1是研究 ActA，细菌表面蛋白，它是唯一的细菌 细菌向宿主细胞移动所需的蛋白质 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，吸引VASP到ActA。 ActA具有四个寡脯氨酸序列 由氨基酸的延伸分开，（第一个，DFPPPPTDE和 其他3个是相似的，一个只有3个P被I中断）; 因此，脯氨酸在起始处具有芳族基团，并且侧接 带负电荷的残基。 这是关键的VASP绑定 所有4个站点都被认为可以运行，但这尚未得到证实。 使用PCR定点突变和细菌表达， PI建议通过凝胶过滤和分析研究结合 离心以鉴定一级结构决定簇 和ActA与VASP之间的复合物的结合常数。 寡脯氨酸类似物的制备和研究， 复合物形成的竞争性抑制剂。 目的2结合了两个目的：黏着斑蛋白与VASP和ActA的结合 在体内发挥作用。 李斯特菌必须吸引VASP 粘着斑蛋白结合在宿主体内 因此，在体外 结合研究将确定VASP与 黏着斑蛋白与ActA。 肽类似物抑制的等级顺序 还将研究ActA-VASP和曲马豆素-ActA结合的影响。 通过显微注射和延时视频显微镜观察。 李斯特菌 ActA突变体将用穿梭载体PMK 4制备， 同源重组与定义的变化， 寡脯氨酸重复序列。 每一个变种人的能力 细菌产生肌动蛋白尾巴，在内部移动并从 细胞间的相互作用以及在小鼠中引起疾病的研究。 目的3：研究VASP与profilin的结合， 该复合物与ActA的相互作用。 这个想法是， 与VASP或VASP-ActA结合可改变profilin 增强肌动蛋白单体上的ATP/ADP交换。

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.