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

描述（由申请人提供）：肠出血性大肠杆菌（EHEC）是食源性细菌病原体，可引起严重和偶尔致命的出血性结肠炎（HC）。幼儿不成比例地受到疾病严重表现的影响，并可能发展为溶血性尿毒症综合征（HUS），这是一种迅速进展且有时致命的肾衰竭形式。溶血性尿毒综合征是由肠出血性大肠杆菌引起的一种罕见并发症，但由于其严重程度、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。肠出血性大肠杆菌被列为CDC/NIAID B类生物危害剂。当溶血性尿毒综合征发生时，通常是在腹泻期恢复后。在肠出血性大肠杆菌诊断时进行的强化液体治疗已显示对溶血性尿毒综合征的发展有一定的保护作用，但治疗仍然是非特异性的，决定溶血性尿毒综合征是否发展的因素尚不完全清楚。HC和HUS都是由志贺毒素（Stx）引起的，在大多数肠出血性大肠杆菌菌株中，志贺毒素在噬菌体上编码，并在噬菌体诱导后释放。Stx的产生和释放的控制是在噬菌体基因组中编码的，但体内控制Stx产生和释放的因素尚不清楚。我们假设，在腹泻期抑制诱导和/或噬菌体功能将抑制体内Stx并与支持治疗协同作用，以改善或预防溶血性尿毒综合征的发生。本提案的目标是确定有助于体内产生Stx的噬菌体功能，因此可能代表体内抑制Stx的靶标。我们将使用；基于噬菌体的重组系统，重组，以产生特定噬菌体基因的突变，并在体外和体内评估编码功能在Stx产生和释放中的作用。两个具体目的是：目的1：确定噬菌体介导的裂解、切除、循环和复制控制Stx2产生和释放的程度。目的2：确定目的1中鉴定的噬菌体功能中哪一种对志贺毒素依赖疾病动物模型的毒力有贡献。

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.