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，可导致脑膜炎、脑膜脑炎、败血症、堕胎，在某些情况下还可导致胃肠炎。总体死亡率>20%，胎儿或新生儿感染李斯特菌的死亡率甚至更高。李斯特菌侵入广泛的细胞类型。胞内李斯特菌在宿主细胞的细胞质中复制，并使用细菌蛋白ActA在细菌表面诱导宿主肌动蛋白丝（“肌动蛋白尾”）的聚合。基于肌动蛋白的运动性允许李斯特菌在细胞间传播而不离开保护性细胞内小生境，并且对于发病机理是必不可少的。然而，李斯特菌运动和传播的机制仍然难以捉摸。衔接蛋白SH 2- Bbeta调节细胞运动。我认为SH 2- Bbeta与李斯特菌的运动性有关。初步数据显示，在过表达野生型SH 2-B β的细胞中的李斯特菌表现出增加的速度（对照的225%），而在宿主细胞中表达SH 2-B β的SH 2结构域缺陷突变体抑制李斯特菌运动（约60%）。在使用爪蟾卵细胞提取物和纯化的GST-SH 2-B β的无细胞系统中，SH 2-Ba使李斯特菌的速度增加了对照的140%。我已经表明，SH 2- Bbeta结合VASP/profilin，这两种蛋白质已被证明参与肌动蛋白依赖性李斯特菌运动。本申请测试了SH 2- Bbeta通过刺激基于肌动蛋白的运动性促进李斯特菌感染的假设。第一个目标将确定VASP/ profilin是否直接结合SH 2- Bbeta。第二个目标将确定SH 2- 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SH 2- Ba是否是李斯特菌感染传播所必需的。 第四个目标是研究SH 2- Bbeta是否是李斯特菌毒力所必需的。除了提供深入了解SH 2- Bbeta有助于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.