The role of prophage life cycle in Stx production and disease due to EHEC

描述（由申请方提供）：肠出血性大肠杆菌（EHEC）是食源性细菌病原体，可引起重度和偶尔致命的出血性结肠炎（HC）。幼儿不成比例地受到严重疾病表现的影响，并可能发展为溶血性尿毒症综合征（HUS），这是一种快速进展的，有时是致命的肾衰竭形式。溶血性尿毒综合征是肠出血性大肠杆菌引起的一种罕见的疾病并发症，但由于其严重性、影响幼儿的倾向以及缺乏任何具体的治疗或预防措施，其重要性被放大。肠出血性大肠杆菌被列为CDC/NIAID B类生物危害因子。 当HUS确实发生时，通常是在从血液流变学阶段恢复之后。在诊断出肠出血性大肠杆菌时开始的强化液体治疗已被证明对HUS的发展具有一定的保护作用，但治疗仍然是非特异性的，并且决定HUS是否发展的因素尚未完全了解。HC和HUS均由滋贺毒素（Stx）引起，在大多数EHEC菌株中，志贺毒素在原噬菌体上编码并在原噬菌体诱导后释放。在噬菌体基因组内编码Stx产生和释放的控制，但是控制体内Stx产生和释放的因素的特征很差。 我们假设，抑制诱导和/或噬菌体的功能，在消化道阶段将抑制在体内的Stx和协同与支持治疗，以改善或预防发病的HUS。该提案的目标是鉴定有助于体内Stx产生的噬菌体功能，因此可以代表体内Stx抑制的靶点。我们将使用;利用基于噬菌体的重组系统，重组工程，在特定的噬菌体基因中产生突变，并评估编码的功能在Stx产生和体外和体内释放中的作用。这两个具体目标是：目标1：确定噬菌体介导的裂解、切除、环化和复制控制Stx 2产生和释放的程度。目标二：确定目标1中鉴定的噬菌体功能中的哪一种有助于在充分表征的滋贺毒素依赖性疾病动物模型中的毒力。

DESCRIPTION (provided by applicant): Enterohemorrhagic Escherichia coli (EHEC) are food-borne bacterial pathogens that cause severe and occasionally fatal hemorrhagic colitis (HC). Young children are disproportionately affected by severe manifestations of disease, and may develop hemolytic-uremic syndrome (HUS), a rapidly progressive and sometimes fatal form of renal failure. HUS is an uncommon complication of disease due to EHEC, but its importance is amplified by its severity, tendency to affect young children, and the absence of any specific treatment or preventative measure. EHEC is listed as a CDC/NIAID category B biohazard agent. When HUS does occur, it is often after recovery from the diarrheic phase. Intensive fluid therapy instituted at the time of EHEC diagnosis has shown to be somewhat protective against HUS development, but therapy remains non-specific and the factors that determine whether or not HUS develops are incompletely understood. Both HC and HUS are caused by Shiga toxins (Stx), which in most EHEC strains are encoded on prophages and released following prophage induction. Control of Stx production and release is encoded within the phage genome, but the factors that control Stx production and release in vivo are poorly characterized. We hypothesize that suppressing induction and/or phage functions during the diarrheic phase will suppress Stx in vivo and synergize with supportive therapy, to ameliorate or prevent the onset of HUS. The goal of this proposal is to identify phage functions that contribute to Stx production in vivo, and therefore could represent targets for Stx suppression in vivo. We will use the ; phage-based recombination system, recombineering, to generate mutations in specific phage genes, and evaluate the role of the encoded functions in Stx production and release both in vitro and in vivo. The two specific aims are: Aim 1: Determine the extent to which phage-mediated lysis, excision, circularization, and replication control Stx2 production and release. Aim 2: Determine which of the phage functions identified in aim 1 contribute to virulence in a well- characterized Shiga toxin-dependent animal model of disease.