Role of Adapter Protein in Infectious Diseases

描述（由申请人提供）：单核细胞增生李斯特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肠胃炎。李斯特菌的总死亡率为20%，胎儿或新生儿感染李斯特菌的死亡率甚至更高。李斯特菌侵入范围广泛的细胞类型。胞内李斯特菌在宿主细胞的细胞质中复制，并利用细菌蛋白ActA诱导宿主肌动蛋白丝（“肌动蛋白尾”）在细菌表面聚合。肌动蛋白为基础的运动性允许李斯特菌在不离开保护性细胞内生态位的情况下从一个细胞扩散到另一个细胞，这对发病至关重要。然而，李斯特菌运动和传播的机制仍然难以捉摸。适配蛋白SH2- Bbeta调节细胞运动。我认为SH2- β与李斯特菌的活力有关。初步数据显示，在过表达野生型SH2- Bbeta的细胞中，李斯特菌的运动速度增加（为对照的225%），而在宿主细胞中表达SH2结构域缺陷突变体SH2- Bbeta抑制李斯特菌的运动（约60%）。在使用爪蟾卵提取物和纯化GST-SH2-Bbeta的无细胞系统中，SH2-Ba使李斯特菌的速度提高了140%。我已经证明SH2- β与VASP/profilin两种蛋白结合，这两种蛋白已被证明参与了动作蛋白依赖性李斯特菌的运动。这一应用验证了SH2- Bbeta通过刺激肌动蛋白为基础的运动促进李斯特菌感染的假设。第一个目的是确定VASP/ profilin是否直接与SH2- Bbeta结合。第二个目的是确定SH2- Bbeta与VASP/profilin的相互作用是否是李斯特菌运动所必需的。第三个目标是测试SH2- Ba是否是李斯特菌感染传播所必需的。第四个目标将检验SH2- Bbeta是否为李斯特菌毒力所必需。除了深入了解SH2- Bbeta促进李斯特菌运动的分子机制外，应用研究的结果将增加我们对李斯特菌传播的基本机制的理解。这些旨在鉴定参与李斯特菌运动的新蛋白和信号通路的研究可能确定新的治疗靶点，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害。

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.