Shiga Toxin Production and Role in Pathogenesis

描述(申请人提供)：大肠杆菌O157：H7是一种新出现的重要病原体。由O157：H7大肠杆菌引起的疾病的特征是腹泻、出血性结肠炎和潜在的致命并发症-溶血性尿毒症综合征(HUS)。志贺毒素(STX)是O157：H7的主要毒力因子。志贺毒素的两个主要抗原变异体STX1和Stx2有55%的氨基酸同源性，然而Stx2的产生与包括HUS在内的严重疾病的进展有关。我们建议研究志贺毒素在大肠杆菌O157引起的疾病发病机制中的两个方面：H7-肠道志贺毒素产生的调节以及STX1和STX2效力差异的分子基础。志贺毒素的基因编码在细菌病毒上。毒素产生的调节与影响病毒生命周期的因素密切相关，特别是诱导病毒裂解周期的因素，例如环丙沙星等抗生素。在特定的目标1中，我们将表征在体外和体内影响Stx2表达的因素。除了毒素的产生量外，感染过程中产生的志贺毒素的类型也会影响疾病的严重程度，Stx2是目前为止效力更强的毒素。虽然很明显Stx2可以诱导细胞死亡，但尚不清楚HUS的发生是否需要细胞死亡，毒性可能发生在机体水平。在特定的目标2中，我们将利用体外细胞培养模型来表征杂合Stx2/STX1突变体，以确定为什么Stx2比STX1更有效。在具体目标3中，我们将使用小鼠疾病模型在体内确定杂交突变体的效力。了解影响志贺毒素产生和效力的病毒、细菌和宿主因素可能会导致减少致命疾病发生率的治疗。

DESCRIPTION (provided by applicant): E. coli O157:H7 is an emerging pathogen of major importance. Disease caused by E. coli O157:H7 is characterized by diarrhea, hemorrhagic colitis, and the potentially fatal complication, hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is a major virulence factor of E. coli O157:H7. Two major antigenic variants of Shiga toxin, Stx1 and Stx2, share 55% amino acid homology, however Stx2 production has been associated with progression to severe disease, including HUS. We propose to examine two aspects of Shiga toxin in the pathogenesis of disease caused by E. coli O157:H7 - regulation of Shiga toxin production in the intestine and the molecular basis for the difference in potency between Stx1 and Stx2. The genes for Shiga toxin are encoded on bacterial viruses. Regulation of toxin production is strongly linked to factors that affect the virus life cycle, specifically factors that induce the viral lytic cycle, for example antibiotics such as ciprofloxicin. In Specific Aim 1 we will characterize factors that influence Stx2 expression in vitro and in vivo. In addition to amount of toxin production, the type of Shiga toxin produced during infection can influence the severity of disease, with Stx2 being by far the more potent toxin. While it is clear that Stx2 can induce cellular death, it is not clear that cellular death is required for development of HUS, and toxicity may occur at the organismal level. In Specific Aim 2 we will characterize hybrid Stx2/Stx1 mutants to determine why Stx2 is more potent than Stx1 using in vitro cell culture models. In Specific Aim 3 we will determine the potency of the hybrid mutants in vivo using a mouse model of disease. Understanding the viral, bacterial and host factors that influence Shiga toxin production and potency could lead to treatments that reduce the incidence of fatal disease.