Mechanisms and significance of intestinal cell proliferation in Vibrio parahaemolyticus infection

项目总结 副溶血性弧菌是引起海产品传播的腹泻病的主要原因，是一种多重耐药菌。 新出现的病原体。尽管已知其中一种3型分泌系统(T3SS2)在 在细菌定植和腹泻疾病中的作用，功能T3SS2装置的组装和 在感染过程中负责病理变化的效应器还没有完全定义。续 这一缺口的存在代表着一个重要的问题，因为在它被填补之前， 通过靶向特定毒力因子的药物干预副溶血性弧菌感染是遥远的。 长期目标是通过定义T3SS2设备组件来利用所提供的医疗好处 以及效应蛋白在副溶血性弧菌感染过程中的作用。这里的总体目标是 确定组装功能性T3SS2装置和增强肠道细胞所需的蛋白质 扩散。中心假设是VopI，功能T3SS2组装的重要组成部分 也是一种效应蛋白，调节肠道细胞的增殖，有利于肠道 殖民主义。这一假设是基于我们自己的初步数据提出的，即删除 VopI阻断T3SS2底物的分泌和转运，VopI本身可以通过 T3SS2进入宿主细胞核，调节细胞增殖和细菌定植。该计划的基本原理 建议的研究是，一旦VopI在T3SS2装置和上皮细胞的组装中发挥作用 增殖被阐明，T3SS2组装和肠上皮细胞增殖可能是 药物调节，导致新的和创新的方法来预防和治疗 感染副溶血性弧菌。此外，这项研究的结果将为 T3SS装置蛋白在体外和体内细菌感染中的效应作用指导原则 强大的初步数据，这一假说将通过追求三个具体目标来检验：1)确定VopI的作用 作为T3SS2设备组件中的结构部件；2)定义VopI、AS 作为效应器，促进细胞增殖；以及3)在婴儿体内确定VopI作为效应器的作用 兔子模型。在第一个目标中，我们将确定VopI本地化及其在T3SS2中的交互伙伴 T3SS2生物发生的电子显微镜、共聚焦显微镜和扫描电子显微镜研究 生化方法。在第二个目标中，我们将阐明Vopi介导的细胞的机制 扩散。特别是，我们将确定Vopi和a之间相互作用的生物学意义。 宿主核仁蛋白，EBP2。在第三个目标中，我们将确定VopI作为效应器在肠道细胞中的作用 体内增殖和细胞增殖对细菌定植和毒力的贡献。

PROJECT SUMMARY Vibrio parahaemolyticus is a leading cause of seafood-borne diarrheal disease and a multidrug resistant emerging pathogen. Although it is known that one of the type 3 secretion systems (T3SS2) plays an essential role in bacterial colonization and diarrheal disease, the assembly of a functional T3SS2 apparatus and the effectors that are responsible for pathological alterations during infection are not completely defined. Continued existence of this gap represents an important problem because, until it is filled, the likelihood that pharmacological intervention of V. parahaemolyticus infection by targeting specific virulence factors is remote. The long-term goal is to harness the medical benefits that are offered by defining T3SS2 apparatus assembly and the function of effector proteins during V. parahaemolyticus infection. The overall objective here is to identify proteins that are required for the assembly of functional T3SS2 apparatus and enhancing intestinal cell proliferation. The central hypothesis is that VopI, an essential component for the assembly of functional T3SS2 apparatus, also serves as an effector protein to regulate intestinal cell proliferation for the benefit of intestinal colonization. This hypothesis has been formulated on the basis of our own preliminary data that deletion of VopI blocked the secretion and translocation of T3SS2 substrates and VopI itself can be translocated by T3SS2 into host cell nucleus to regulate cell proliferation and bacterial colonization. The rationale for the proposed research is that, once the role of VopI in the assembly of T3SS2 apparatus and epithelial cell proliferation is elucidated, both T3SS2 assembly and intestinal epithelial cell proliferation could be pharmacologically modulated, resulting in new and innovative approaches for the prevention and treatment of infection with V. parahaemolyticus. Furthermore, the results obtained from this study will shed new light on the role of T3SS apparatus protein as an effector both in vitro and in vivo during bacterial infection. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Define the role of VopI as a structural component in the assembly of T3SS2 apparatus; 2) Define the mechanisms by which VopI, as an effector, promotes cell proliferation; and 3) Define the role of VopI as an effector in vivo using an infant rabbit model. In the first aim, we will determine VopI localization, its interaction partners within the T3SS2 apparatus and its effect on T3SS2 biogenesis using electron microscopy, confocal microscopy and biochemical approaches. In the second aim, we will elucidate the mechanism of VopI-mediated cell proliferation. Particularly, we will determine the biological significance of the interaction between VopI and a host nucleolar protein, EBP2. In the third aim, we will determine the role of VopI, as an effector, in intestinal cell proliferation in vivo and the contribution of cell proliferation to bacterial colonization and virulence.