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

描述(申请人提供)：肠出血性大肠杆菌(EHEC)是一种食源性细菌病原体，可导致严重的、有时甚至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)，这是一种进展迅速、有时甚至致命的肾功能衰竭形式。HUS是EHEC引起的一种罕见的疾病并发症，但由于其严重性、易影响幼儿以及缺乏任何特定的治疗或预防措施，其重要性被放大。EHEC被列为CDC/NIAID B类生物危害剂。当HUS确实发生时，通常是在从腹泻阶段恢复之后。在EHEC确诊时开始的强化液体治疗对HUS的发展有一定的保护作用，但治疗仍然没有特异性，决定HUS是否发展的因素还不完全清楚。HC和HUS都是由志贺毒素(STX)引起的，在大多数EHEC菌株中，志贺毒素(STX)编码在前噬菌体上，并在前噬菌体诱导后释放。控制STX的产生和释放是在噬菌体基因组中编码的，但在体内控制STX产生和释放的因素尚不清楚。我们假设，在腹泻阶段抑制诱导和/或噬菌体功能将抑制体内的STX，并与支持性治疗协同作用，以改善或防止HUS的发生。这项提议的目的是确定在体内促进STX产生的噬菌体功能，从而可能成为体内抑制STX的靶点。我们将使用基于噬菌体的重组系统，重组工程，来产生特定噬菌体基因的突变，并评估编码功能在体外和体内STX生产和释放中的作用。两个特定的目标是：目标1：确定噬菌体介导的裂解、切除、环化和复制控制Stx2的产生和释放的程度。目的2：确定目标1中确定的哪些噬菌体功能与志贺毒素依赖疾病动物模型的毒力有关。 公共卫生相关性：肠出血性大肠杆菌(EHEC)是人类的食源性细菌病原体，可导致出血性结肠炎，有时还会导致溶血性尿毒症综合征(HUS)，这是一种进展迅速、往往致命的肾功能衰竭，最常影响儿童。HUS是EHEC引起的一种罕见的疾病并发症，但其重要性因其严重性、影响幼儿的倾向以及缺乏任何特定治疗或预防措施而变得更加重要。该项目的目标是确定新的治疗靶点，以预防或改善由于EHEC引起的HUS。

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. PUBLIC HEALTH RELEVANCE: Enterohemorrhagic Escherichia coli (EHEC) are food-borne bacterial pathogens of humans that cause hemorrhagic colitis and occasionally hemolytic-uremic syndrome (HUS), a rapidly progressive often fatal form of renal failure that most often affects children. HUS is an uncommon complication of disease due to EHEC, but its importance is amplified by its severity, its tendency to affect young children, and the absence of any specific treatment or preventative measure. The goal of this project is to identify new targets for therapy to prevent or ameliorate HUS due to EHEC.