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（由申请人提供）：单核细胞增生李斯特氏菌是一种食源性病原体，可引起脑膜炎、脑膜脑炎、败血症、流产，在某些情况下还可引起胃肠炎。总死亡率>20%，胎儿或新生儿感染李斯特菌的死亡率更高。李斯特菌侵入多种细胞类型。细胞内李斯特菌在宿主细胞的细胞质中复制，并使用细菌蛋白 ActA 诱导宿主肌动蛋白丝（“肌动蛋白尾部”）在细菌表面聚合。基于肌动蛋白的运动使李斯特菌能够在不离开保护性细胞内生态位的情况下从一个细胞传播到另一个细胞，这对于发病机制至关重要。然而，李斯特菌运动和传播的机制仍然难以捉摸。接头蛋白 SH2-Bbeta 调节细胞运动。我认为 SH2-Bbeta 与李斯特菌的运动有关。初步数据显示，过表达野生型 SH2-Bbeta 的细胞中的李斯特菌表现出增加的速度（对照的 225%），而宿主细胞中 SH2-Bbeta 的 SH2 结构域缺陷突变体的表达抑制李斯特菌运动（约 60%）。在使用非洲爪蟾卵细胞提取物和纯化的 GST-SH2-Bbeta 的无细胞系统中，SH2-Ba 使李斯特菌的生长速度比对照增加了 140%。我已经证明 SH2-Bbeta 与 VASP/profilin 两种蛋白结合，这两种蛋白已被证明参与肌动蛋白依赖性李斯特菌运动。该应用测试了 SH2-Bbeta 通过刺激基于肌动蛋白的运动来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 与 VASP/profilin 的相互作用是否是李斯特菌运动所必需的。第三个目标将测试SH2-Ba是否是李斯特菌感染传播所必需的。 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加深我们对李斯特菌传播基本机制的理解。这些旨在识别与李斯特菌运动有关的新蛋白质和信号通路的研究可能会确定新的治疗靶点，以防止李斯特菌感染的快速传播，从而保护人们免受李斯特菌病的侵害。