Role of Adapter Protein in Infectious Diseases

衔接蛋白在传染病中的作用

• 批准号: 6820988
• 负责人: MARIA DIAKONOVA
• 金额: $ 30.53万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 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 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6820988
• 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(申请人提供)：单核细胞增多性李斯特菌是一种食源性病原体，可导致脑膜炎、脑膜脑炎、败血症、流产，在某些情况下还会导致胃肠炎。总的死亡率为20%，胎儿或新生儿感染李斯特菌的死亡率更高。李斯特菌侵入多种细胞类型。李斯特菌在宿主细胞的细胞质内复制，并利用细菌蛋白ActA诱导宿主肌动蛋白细丝(“肌动蛋白尾巴”)在细菌表面聚合。基于肌动蛋白的运动使李斯特菌能够在细胞之间传播，而不离开保护性的细胞内生态位，这在致病过程中是必不可少的。然而，李斯特菌运动和传播的机制仍然不清楚。适配蛋白SH2-Bbeta调节细胞的运动。我已经将SH2-Bbeta与李斯特菌的运动联系在一起。初步数据显示，在过量表达野生型SH2-Bβ的细胞中，李斯特菌的移动速度加快(对照的225%)，而SH2-Bβ的SH2结构域缺陷突变体在宿主细胞中的表达抑制了李斯特菌的移动(约60%)。在使用非洲爪哇提取物和纯化的GST-SH2-Bβ的无细胞系统中，SH2-BA使李斯特氏菌的生长速度提高了140%。我已经证明了SH2-Bbeta与Vasp/profilin结合，这两种蛋白已被证明参与依赖肌动蛋白的李斯特菌的运动。这一应用验证了SH2-Bbeta通过刺激基于肌动蛋白的运动性而促进李斯特菌感染的假设。第一个目的将确定VASP/Profilin是否直接与SH2-Bβ结合(S)。第二个目的是确定SH2-Bbeta是否与Vasp/profilin相互作用是李斯特菌运动所必需的。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是否是李斯特菌毒力所必需的。除了深入了解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李斯特菌传播的基本机制的理解。这些研究旨在识别与李斯特菌运动有关的新蛋白和信号通路，可能会发现新的治疗靶点，以防止李斯特菌感染的快速分布，从而保护人们免受李斯特菌病的侵袭。