Mechanisms and significance of intestinal cell proliferation in Vibrio parahaemolyticus infection

 描述（由申请方提供）：副溶血性弧菌是海产品传播性腹泻病的主要原因，也是一种多重耐药的新兴病原体。尽管已知3型分泌系统之一（T3SS2）在细菌定殖和结肠炎疾病中起重要作用，但在感染期间负责病理改变的功能性T3SS2装置和效应器的组装并不完全确定。这一差距的持续存在是一个重要问题，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T3SS2装置组装和副溶血性弧菌感染期间效应蛋白的功能所提供的医疗益处。本文的总体目标是鉴定功能性T3SS2装置组装和增强肠细胞增殖所需的蛋白质。中心假设是，VopI，功能T3SS2装置的组装的重要组成部分，也作为一种效应蛋白来调节肠细胞增殖的肠道定植和毒力的好处。这一假设是基于我们自己的初步数据，即VopI的缺失阻断了T3SS2底物的分泌和转运，并且VopI本身可以通过T3SS2转运到宿主细胞核中以调节细胞增殖和细菌定殖。的理由 所提出的研究是，一旦阐明了VopI在T3SS2装置组装和上皮细胞增殖中的作用，T3SS2组装和肠上皮细胞增殖都可以被间接调节，从而产生预防和治疗副溶血性弧菌感染的新的和创新的方法。此外，从这项研究中获得的结果将揭示新的光T3SS装置蛋白的作用，在体外和体内的细菌感染过程中的效应。在强有力的初步数据的指导下，这一假设将通过追求三个具体目标进行测试：1）定义VopI作为功能性T3SS2装置组装中的重要组分的作用; 2）定义VopI作为效应物促进细胞增殖的机制; 3）使用幼兔模型定义VopI作为体内效应物的作用。在第一个目标中，我们将使用电子显微镜和生物化学方法确定VopI定位及其在T3SS2装置内的相互作用伙伴。在第二个目标中，我们将阐明VopI介导的细胞增殖的机制。特别是，我们将确定VopI和宿主核仁蛋白EBP 2之间相互作用的生物学意义。在第三个目标中，我们将确定VopI作为效应子在体内肠细胞增殖中的作用以及细胞增殖对细菌定植和毒力的贡献。

 DESCRIPTION (provided by applicant): 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 and virulence. This hypothesis has been formulated on the basis of our own preliminary data that 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 an essential component in the assembly of a functional 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 and its interaction partners within the T3SS2 apparatus using electron 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 ell proliferation to bacterial colonization and virulence.