Role of Adapter Protein in Infectious Diseases

衔接蛋白在传染病中的作用

• 批准号: 7407303
• 负责人: MARIA DIAKONOVA
• 金额: $ 4.89万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7407303
• 关键词: Listeria; Listeria infections; SDS polyacrylamide gel electrophoresis; Salmonella; actin binding protein; bacteria infection mechanism; bacterial proteins; binding proteins; cell motility; genetically modified animals; green fluorescent proteins; laboratory mouse; molecular site; pathologic process; protein binding; protein structure function; virulence; western blottings

DESCRIPTION (provided by applicant): Listeria monocytogenes is a food-borne pathogen that can cause meningitis, meningoencephalitis, septicemias, abortions and, in some cases, gastroenteritis. The overall mortality rate is >20% and fetal or neonatal infection with Listeria has an even higher mortality. Listeria invades a broad range of cell types. Intracellular Listeria replicates in the cytoplasm of host cells and induces the polymerization of host actin filaments ("actin tails") at the bacteria surface using bacterial protein ActA. Actin-based motility allows Listeria to spread from cell to cell without leaving the protective intracellular niche, and is essential for pathogenesis. However, the mechanism underlying Listeria motility and spreading remains elusive. The adapter protein SH2- Bbeta regulates cell motility. I have implicated SH2- Bbeta in the motility of Listeria. Preliminary data revealed that Listeria in cells overexpressing wild type SH2- Bbeta demonstrates increased velocity (225% of control) while expression of SH2 domain-deficient mutants of SH2- Bbeta in host cells inhibits Listeria movement (by approximately 60%). In a cell-free system using Xenopus ooeyte extracts and purified GST-SH2-Bbeta, SH2-Ba increased the velocity of Listeria by 140% of control. I have shown that SH2- Bbeta binds to VASP/profilin two proteins that have been shown to participate in actin-dependent Listeria motility. This application tests the hypothesis that SH2- Bbeta promotes Listeria infection by stimulating actin-based motility. The first aim will determine whether VASP/ profilin directly bind(s) to SH2- Bbeta. The second aim will determine whether SH2- Bbeta interaction with VASP/profilin is required for Listeria motility. The third aim will test whether SH2- Ba is required for spreading of Listeria infection. The fourth aim will examine whether SH2- Bbeta is required for the virulence of Listeria. In addition to providing insight into the molecular mechanism by which SH2- Bbeta contributes to Listeria motility, the results of the application studies will increase our understanding of the fundamental mechanism by which Listeria spreads. These studies designed to identify new proteins and signaling pathways involved in Listeria motility may identify new therapeutic targets for preventing the rapid distribution of Listeria infection and thereby protect people from listeriosis.

描述（由申请人提供）：单核细胞增生李斯特菌是一种食物传播的病原体，可引起脑膜炎，脑膜脑炎，败血症，流产，在某些情况下是胃肠炎。总体死亡率> 20％，胎儿或新生儿感染李斯特菌的死亡率更高。李斯特菌入侵了广泛的细胞类型。细胞内李斯特菌在宿主细胞的细胞质中复制，并诱导使用细菌蛋白ACTA在细菌表面的宿主肌动蛋白丝（肌动蛋白尾巴）的聚合。基于肌动蛋白的运动允许李斯特菌在细胞到细胞中扩散，而不会留下保护性细胞内的小裂，对于发病机理至关重要。但是，李斯特菌运动和扩散的基础机制仍然难以捉摸。衔接蛋白SH2- BBETA调节细胞运动。我已将SH2-BBETA牵涉到李斯特菌的运动。初步数据表明，过表达野生型SH2-BBETA的细胞中的李斯特菌表现出速度增加（对照的225％），而SH2结构域缺陷型sh2- bbeta的表达抑制了李斯特菌运动（大约60％）。在使用异爪蟾植物提取物和纯化的GST-SH2-BBETA的无细胞系统中，SH2-BA将李斯特菌的速度提高了140％的对照。我已经表明，SH2-BBETA与VASP/profilin的两种蛋白结合，已显示出参与肌动蛋白依赖性李斯特菌运动。该应用检验了SH2-BBETA通过刺激基于肌动蛋白的运动促进李斯特菌感染的假设。第一个目标将确定VASP/ profilin是否直接与SH2-BBETA结合。第二个目标将确定SH2-BBETA是否与李斯特菌运动需要与VASP/Profilin的相互作用。第三个目标将测试是否需要SH2-BA来传播李斯特菌感染。 第四个目标将检查SH2-BBETA是否需要李斯特菌的毒力。除了洞悉SH2-BBETA促进李斯特菌运动的分子机制外，应用研究的结果还将增加我们对利斯特菌扩散的基本机制的理解。这些旨在鉴定李斯特菌运动中涉及的新蛋白质和信号通路的研究可能会确定用于防止李斯特菌感染快速分布的新治疗靶标，从而保护人们免受李斯特氏病的影响。