Mechanisms and significance of intestinal cell proliferation in Vibrio parahaemolyticus infection

项目摘要 副溶血性弧菌（Vibrio parahaemolyticus）是引起海产品传播性腹泻病的主要原因， 新兴病原体尽管已知3型分泌系统之一（T3 SS 2）在细胞内起重要作用， 在细菌定植和牙周炎疾病中的作用，功能性T3 SS 2装置的组装和 在感染过程中引起病理改变的效应物还没有完全确定。继续 这一差距的存在是一个重要的问题，因为在填补这一差距之前， 通过靶向特定毒力因子对副溶血性弧菌感染进行药物干预的可能性很小。 长期目标是利用通过定义T3 SS 2装置组件所提供的医疗益处 以及副溶血性弧菌感染过程中效应蛋白的功能。总体目标是 鉴定组装功能性T3 SS 2装置和增强肠细胞增殖所需的蛋白质。 增殖中心假设是VopI，功能性T3 SS 2组装的重要组分， 装置，也作为一种效应蛋白，以调节肠细胞增殖的利益，肠 殖民化这一假设是根据我们自己的初步数据提出的， VopI阻断了T3 SS 2底物的分泌和转运，VopI本身可以通过 T3 SS 2进入宿主细胞核以调节细胞增殖和细菌定殖。的理由 提出的研究是，一旦VopI在T3 SS 2装置和上皮细胞组装中的作用 增殖，T3 SS 2组装和肠上皮细胞增殖都可以被阐明。 制定新的和创新的预防和治疗方法， 副溶血性弧菌感染。此外，从这项研究中获得的结果将揭示新的光 在细菌感染过程中，T3 SS装置蛋白在体外和体内作为效应物的作用。指导 强有力的初步数据，这一假设将通过追求三个具体目标进行检验：1）定义的作用，VOPI 作为T3 SS 2装置组装中的结构部件; 2）定义VopI作为 作为效应子，促进细胞增殖;和3）使用婴儿来定义VopI作为体内效应子的作用 兔子模型在第一个目标中，我们将确定VopI本地化，其在T3 SS 2内的相互作用伙伴 仪器和它对T3 SS 2生物发生的影响，使用电子显微镜，共聚焦显微镜和 生物化学方法。在第二个目标中，我们将阐明VopI介导的细胞凋亡的机制， 增殖特别地，我们将确定VopI和一种新的蛋白质之间相互作用的生物学意义。 宿主核仁蛋白EBP 2。在第三个目标中，我们将确定VopI作为效应子在肠细胞中的作用， 体内增殖以及细胞增殖对细菌定殖和毒力的贡献。

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.