Pathogenicity of Shiga Toxin Producing E.coli

描述(申请人提供)：大肠杆菌O157：H7是美国最常见的出血性腹泻或出血性结肠炎(HC)的感染性原因，而非O157：H7志贺氏菌毒素产生的大肠杆菌(STEC)的发病率约为O157：H7的一半。此外，溶血性尿毒症综合征(HUS)，特别是O157：H7的后遗症，也是其他STEC感染的后遗症，是美国儿童急性肾衰竭最常见的基础。大肠杆菌O157：H7属于STEC的一个亚群，称为肠出血性大肠杆菌(EHEC)，它不仅产生志贺毒素(STX)1型或2型(或其变体)，而且含有pO157(或相关质粒)并表达粘附素内膜蛋白，粘附素内膜蛋白由EAE基因编码，位于一个称为肠细胞消失点的致病岛上。内膜与细菌表达的易位受体TIR结合，也与真核细胞受体核仁素结合，尽管结合的程度较低。我们最近发现，Stx2增加了HEp2细胞表面核仁素的水平，可能是由于这种毒素介导的肠上皮细胞表面的改变，表达Stx2的EHEC O157菌株比其同基因Stx2阴性突变株更好地在小鼠肠道定植。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致病的致病机制，并测试预防和治疗这些疾病的策略。其具体目的是：1.研究STX1和Stx2在口服接种荷兰带兔后是否都有助于肾脏损害和/或其他与HUS相关的临床特征；2.检查产生Stx2d-肠粘液激活毒素(Stx2dact)的STEC菌株在口服感染链霉素治疗的小鼠中毒力增强的基础，Stx2dact是由EAE阴性的STEC菌株产生的Stx2变体，已与欧洲患者的血性腹泻和HUS有关；3.继续探讨Stx2在大肠杆菌O157：H7感染小鼠肠道中的作用，检测Stx2对体内核仁表达的影响，并检测Stx1是否能增强O157：H7的定植；以及4.确定Stx2是否调节上皮细胞中核仁的表达和/或分布，以及这种变化是否与STX诱导的细胞凋亡有关。公共卫生相关性：由于O157：H7感染的潜在严重性，以及该生物具有非常低的50%感染量，并且可以在人与人之间传播，因此大肠杆菌O157：H7被归类为B类生物制剂。作为这一项目的成果，我们将进一步描述这种病原体致病的步骤，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.