Mechanisms and significance of intestinal cell proliferation in Vibrio parahaemolyticus infection

 描述(由申请人提供)：副溶血性弧菌是海鲜传播的腹泻疾病的主要原因，也是一种对多种药物产生抗药性的新病原体。尽管已知其中一种3型分泌系统(T3SS2)在细菌定植和腹泻疾病中起着重要作用，但T3SS2装置的功能组装和导致感染过程中病理变化的效应器尚未完全确定。这一缺口的持续存在代表着一个重要的问题，因为在填补缺口之前，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。长期目标是通过确定T3SS2装置组装和副溶血性弧菌感染期间效应蛋白的功能来利用所提供的医疗益处。这里的总体目标是确定组装功能性T3SS2装置和促进肠道细胞增殖所需的蛋白质。核心假设是，VopI是组装T3SS2功能装置的重要组成部分，也是调节肠道细胞增殖的效应蛋白，有利于肠道定植和毒力。这一假说是基于我们自己的初步数据提出的，即VopI的缺失阻止了T3SS2底物的分泌和转位，并且VopI本身可以被T3SS2转位到宿主细胞核中，以调节细胞增殖和细菌定植。其基本原理是 本研究认为，一旦VopI在T3SS2的组装和肠上皮细胞增殖中的作用被阐明，T3SS2的组装和肠上皮细胞的增殖都可以被药物调控，从而为预防和治疗副溶血性弧菌感染开辟新的创新途径。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过程中作为效应器的作用提供新的线索。在强大的初步数据的指导下，这一假说将通过追求三个具体目标来检验：1)确定VopI作为功能性T3SS2装置组装中的关键组件的作用；2)确定VopI作为效应器促进细胞增殖的机制；以及3)使用幼兔模型确定VopI作为效应器在体内的作用。在第一个目标中，我们将使用电子显微镜和生化方法确定VopI在T3SS2装置中的定位及其相互作用伙伴。在第二个目标中，我们将阐明VopI介导的细胞增殖机制。特别是，我们将确定VopI和宿主核仁蛋白EBP2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作为效应分子在体内肠道细胞增殖中的作用，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.