Stx-mediated disease and immunomodulatory effectors of enterohemorrhagic E.coli

描述(申请人提供)：肠出血性大肠杆菌(EHEC)O157：H7是一种重要的病原体，可引起危及生命的疾病。肠出血性大肠杆菌感染的抗生素治疗与更严重的系统性疾病有关。因此，需要更深入地了解这种病原体的毒力特性，以制定新的治疗策略。这种病原体的效力源于两个主要毒力成分的联合活性：STX毒素和一个复杂的III型分泌系统(TTSS)，包括注射到哺乳动物细胞中的相关效应蛋白。虽然这些成分都得到了广泛的研究，但对这两种主要毒力机制如何相互作用和协同作用知之甚少。例如，STX的组织破坏作用被宿主的先天免疫反应大大增强，罗森肖恩博士的实验室(以及其他人)已经表明，一组分泌的III型“免疫调节”效应蛋白下调了宿主的炎症反应。解决免疫调节效应物的影响的一个主要障碍是缺乏一个模型系统来概括依赖TTSS的宿主定植背景下的STX活性。申请者最近开发的两个模型允许进行此类研究。其中一种使用了一种通过基因工程产生STX的轮状芽孢杆菌菌株，这是一种编码TTSS的小鼠病原体，以及一系列与EHEC非常相似的效应器。这种经过基因改造的轮状芽胞杆菌会诱发一种小鼠疾病，就像人类EHEC感染一样，表现为肠道损伤、肾功能障碍、进行性体重减轻和死亡。第二种模型涉及肠出血性大肠杆菌感染SCID小鼠体内的人异种肠道移植，这是一种模拟人类感染的模型，特别是关于肠出血性大肠杆菌对结肠组织的趋向性。 对减少或加剧严重STX介导性疾病的因素的洞察可能导致预防EHEC感染的最致命后果的新的和必要的战略。作为实现这一长期目标的第一步，我们研究了一组重要的效应蛋白，这些蛋白已被证明在体外调节宿主细胞的炎症反应。为了测试体外鉴定的这些效应器的免疫调节活性是否准确地反映了体内的抗炎作用，以及这些效应器蛋白是否影响STX的传递、传播和导致全身疾病的能力，我们将追求以下具体目标： 1.免疫调节效应剂产生缺陷的CR(？stx2dact)和EHEC菌株的构建及体外鉴定 2.上述CR(？stx2dact)菌株对小鼠的定植能力和对STX介导的局部和全身损伤的能力。 3.利用人体肠道异种移植模型表征上述EHEC菌株对STX介导的局部和全身损伤的能力。

DESCRIPTION (provided by applicant): Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important pathogen that causes life-threatening disease. Antibiotic treatment of EHEC infection is associated with more severe systemic disease. Thus, a deeper understanding of the virulence properties of this pathogen is needed to generate new therapeutic strategies. The potency of this pathogen stems from the combined activity of two major virulence components: Stx toxin and a complex type III secretion system (TTSS), including the associated effector proteins that are injected into mammalian cells. While each of these components was studied extensively, little is known on how these two major virulence mechanisms might interact and synergize. For example, the tissue damaging action of Stx is greatly potentiated by the host innate immune response, and Dr. Rosenshine's laboratory (as well as others) has shown that a group of type III-secreted "immunomodulatory" effector proteins down-regulate the inflammatory response of the host. A major obstacle in addressing the influence of immunomodulatory effectors has been the lack of a model system that recapitulates Stx activity in the context of TTSS-dependent colonization of the host. Two models recently developed by the applicants allow such studies. One employs a genetically engineered Stx-producing strain of C. rodentium, a murine pathogen that encodes a TTSS and a collection of effectors very similar to those of EHEC. This engineered C. rodentium induces a murine disease that, like human EHEC infection, features intestinal damage, renal dysfunction, progressive weight loss and death. A second model involves EHEC infection of human intestinal xenografts in SCID mice, a model that mimics human infection, particularly with respect to the documented tropism of EHEC for colonic tissue. Insights into factors that diminish or exacerbate serious Stx-mediated disease may lead to new and needed strategies to prevent the most lethal consequences of EHEC infection. As a first step towards achieving this long-term goal, we investigate an important group of effector proteins that have been shown to modulate the inflammatory response of host cells in vitro. To test whether the immunomodulatory activities of these effectors identified in vitro accurately reflect anti-inflammatory effects in vivo, and whether these effector proteins influence the delivery, dissemination and capacity of Stx to cause systemic disease, we will pursue the following specific aims: 1. Construction and in vitro characterization of CR (?stx2dact) and EHEC strains defective for production of the immunomodulatory effectors. 2. Characterization of the ability of the above set of CR (?stx2dact) strains to colonize mice and cause local and systemic Stx-mediated damage. 3. Characterization of the ability of the above set of EHEC strains to cause local and systemic Stx-mediated damage using the human intestinal xenograft model.