Novel Type IV Pili of Enterohemorrhagic E. coli O157:H7

性状（由申请方提供）：肠出血性大肠杆菌。大肠杆菌O 157：H7是引起人类出血性结肠炎和溶血性尿毒综合征的重要病原体。肠出血性大肠杆菌O 157：H7菌株产生了一种强效的滋贺毒素，与溶血尿毒综合征的发病机制有关。在O 157：H7菌株中尚未可重复地鉴定出皮利，因此，关于皮利是否在其天然牛或偶然人类宿主的肠道定殖中发挥作用仍然是一个谜。我们最近鉴定了EHEC菌株EDL 933和其他O 157：H7菌株产生的新菌毛。这些皮利，在本文中称为出血性大肠杆菌皮利1（Hcp 1），属于毒性相关的IV型皮利家族。此外，纯化的Hcp 1显示兔红细胞的凝集，并结合到纤连蛋白和层粘连蛋白，这表明在结合到人类蛋白质的粘附作用。一个同基因的Hcp 1缺陷型突变体显示出相当低的水平的粘附培养细胞。菌毛蛋白单体由染色体前菌毛蛋白肽酶依赖性ppdD基因编码，该基因与IV型样纤毛形成基因hofBC相邻。根据我们的初步数据，我们假设Hcp 1是一个关键的毒力因子，有助于EHEC的粘附特性。总的来说，本提案的目的是通过阐明EHEC O 157：H7粘附于培养中的人类上皮细胞的机制来推进对EHEC发病机制的认识。本研究将采用分子生物学、细胞生物学、高倍电子显微镜超微结构分析、生物化学和抗原分析等多学科方法，扩展我们目前对EHEC O 157：H7与宿主靶细胞相互作用的认识。这一建议的结果将为检测E。大肠杆菌O 157：H7在食物来源和水库，重要的是预防和控制肠出血性大肠杆菌感染的人。该建议的中心焦点在于EHEC产生的HCP的结构-功能研究。因此，提出了以下具体目标：1）确定Hcp 1生物发生所需的基因; 2）确定Hcp 1的作用; 3）分析ppdD的表达。

DESCRIPTION (provided by applicant): Enterohemorrhagic E. coli (EHEC) O157:H7 is recognized as an important emerging pathogen responsible for producing hemorrhagic colitis and the hemolytic uremic syndrome (HUS) in humans. EHEC O157:H7 strains elaborate a potent Shiga toxin, which has been associated with the pathogenesis of HUS. No pili have yet been reproducibly identified in O157:H7 strains and therefore, it is still an enigma as to whether pili play a role in colonization of the intestine of their natural bovine or accidental human hosts. We have recently identified a novel pilus produced by EHEC strain EDL933 and other O157:H7 strains. These pili, herein called hemorrhagic coli pili 1 (Hcp1), belong to the virulence-associated type IV pili family. Further, purified Hcp1 showed agglutination of rabbit erythrocytes and bound to fibronectin and laminin suggesting an adhesive role in binding to human proteins. An isogenic Hcp1-deficient mutant showed considerably low levels of adherence to cultured cells. The pilin monomer is encoded by the chromosomal prepilin peptidase-dependent ppdD gene, which is adjacent to the type IV-like piliation genes hofBC. Based on our preliminary data, we hypothesize that Hcp1 is a key virulence factor that contributes to the adhesive properties of EHEC. Overall, the objective of this proposal is to advance knowledge of EHEC pathogenesis by elucidating the mechanism(s) of adherence of EHEC O157:H7 to human epithelial cells in culture. Several multidisciplinary approaches involving molecular biology, cell biology, ultrastructural analysis by high power electron microscopy, and biochemical and antigenic analysis will be carried out to extend our current knowledge on the interaction of EHEC O157:H7 with host target cells. The outcome of this proposal will provide important implications for detection of E. coli O157:H7 in food sources and reservoirs and importantly for prevention and control of EHEC infections in humans. The central focus of this proposal lies in structure-function studies of Hcp produced by EHEC. Thus, the following specific aims are proposed: 1) To define the genes required for Hcp1 biogenesis; 2) To define the role of Hcp1; 3) To analyze expression of ppdD.