Pathogenicity of Shiga Toxin Producing E.coli

描述（由申请人提供）：大肠杆菌O 157：H7是美国血性腹泻或出血性结肠炎（HC）的最常见感染原因，非O 157：H7产滋贺毒素大肠杆菌（STEC）的发病率约为O 157：H7的一半。此外，溶血性尿毒综合征（HUS），特别是O 157：H7的后遗症，但也是其他STEC感染，是美国儿童急性肾衰竭最常见的基础。E.大肠杆菌O 157：H7属于肠出血性大肠杆菌。大肠杆菌（EHEC），其不仅产生滋贺毒素（Stx）1型或2型（或其变体），而且还具有pO 157（或相关质粒）并表达粘附素紧密蛋白，所述粘附素紧密蛋白是由包含在称为肠上皮细胞消失位点的致病性岛内的eae基因编码的外膜蛋白。内膜结合其细菌表达的易位受体Tir，也结合，尽管不那么强烈，真核受体核仁素。我们最近发现，Stx 2增加了HEp 2细胞表面的核仁素水平，可能是由于这种毒素介导的肠上皮细胞细胞表面的改变，Stx 2表达EHEC O 157菌株比其同基因Stx 2阴性突变体更好地定殖小鼠肠道。该项目的长期目标是在分子、细胞和整个动物水平上确定STEC引起疾病的致病机制，并测试预防和治疗这些疾病的策略。具体目标是：1.研究Stx 1和Stx 2是否都有助于肾脏病变的发展和/或其他HUS相关的临床特征时，产生于相同的E. coliO 157：H7菌株经口接种荷兰白兔后，一种新的HUS模型; 2.检查产生Stx 2d-肠粘液可激活毒素（Stx 2dact）的STEC菌株在经口感染、链霉素处理的小鼠中增强的毒力的基础，Stx 2dact是一种Stx 2变体，当由eae阴性STEC菌株产生时，其与欧洲患者的血性腹泻和HUS相关; 3.进一步探讨Stx 2在E. coli O 157：H7在小鼠肠道中的定植，测量Stx 2对体内核仁素表达的影响，并测试Stx 1是否可以增强O 157：H7定植;以及，4.确定Stx 2是否调节上皮细胞中核仁素的表达和/或分布，以及这些变化是否与Stx诱导的中毒细胞凋亡有关。公共卫生相关性：由于O 157：H7感染的潜在严重性，并且由于该生物体具有非常低的50%感染剂量，并且可以在人与人之间传播，因此大肠杆菌O 157：H7被分类为B类生物制剂。作为该项目的成果，我们将进一步描述这种病原体引起疾病的步骤，这反过来可能有助于制定更有针对性的策略来干扰感染和中毒过程。

DESCRIPTION (provided by applicant): Escherichia coli O157:H7 is the most common infectious cause of bloody diarrhea, or hemorrhagic colitis (HC), in the U.S, and the incidence of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) is about half that of O157:H7. Moreover, the hemolytic uremic syndrome (HUS), a sequela of O157:H7 in particular but also other STEC infection, is the most frequent basis for acute kidney failure in U.S. children. E. coli O157:H7 belongs to a subset of STEC called Enterohemorrhagic E. coli (EHEC) that not only make Shiga toxin (Stx) type 1 or type 2 (or a variant thereof) but also harbor pO157 (or a related plasmid) and express the adhesin intimin, an outer membrane protein encoded by the eae gene contained within a pathogenicity island called the locus of enterocyte effacement. Intimin engages its bacterially-expressed translocated receptor Tir and also binds, albeit less avidly, to the eukaryotic receptor nucleolin. We recently found that Stx2 increased the level of nucleolin on the surface of HEp2 cells and, possibly as a consequence of such a toxin-mediated alteration of the cell surface of enterocytes, an Stx2-expressing EHEC O157 strain colonized the intestines of mice better than did its isogenic Stx2-negative mutant. The long-term goals of this project are to define at the molecular, cellular, and whole animal levels the pathogenic mechanisms by which STEC cause disease and to test strategies to prevent and treat these illnesses. The specific aims are to: 1. investigate whether both Stx1 and Stx2 contribute to the development of kidney lesions and/or other HUSrelated clinical features when produced from the same E. coli O157:H7 strain after oral inoculation of Dutch belted rabbits, a newly described model of HUS; 2. examine the basis for the enhanced virulence in orallyinfected, streptomycin-treated mice of STEC strains that produce the Stx2d-intestinal mucus activatable toxin (Stx2dact), an Stx2 variant that when produced by eae-negative STEC strains has been associated with bloody diarrhea and HUS in European patients; 3. continue to probe the role of Stx2 in the establishment of infection by E. coli O157:H7 in the murine gut, measure the impact of Stx2 on nucleolin expression in vivo, and test whether Stx1 can augment O157:H7 colonization; and, 4. determine whether Stx2 modulates nucleolin expression and/or distribution in epithelial cells and whether such changes are linked to Stx-induced apoptosis of intoxicated cells. Public health relevance: Due to the potential severity of O157:H7 infection and because the organism has a very low 50% infectious dose and can spread from person to person, E.coli O157:H7 is classified as a category B biological agent. As an outcome of this project, we will further delineate the steps by which this pathogen causes disease, which, in turn, may facilitate the creation of more targeted strategies to interfere with the infectious and intoxication processes.