Novel YcbQ Pili of Enterohemorrhagic E. coli O157:H7

描述(申请人提供)：肠出血性大肠杆菌(EHEC)O157：H7被认为是一种重要的新兴病原体，可导致人类出血性结肠炎和溶血性尿毒症综合征(HUS)。EHEC O157：H7菌株产生一种有效的志贺毒素，这种毒素与HUS的发病机制有关。到目前为止，还没有在O157：H7菌株中发现菌毛，因此，菌毛是否在其自然的牛或意外的人类宿主的肠道定植中起作用仍是一个谜。我们最近鉴定和纯化了EHEC菌株EDL933和其他O157：H7菌株产生的一种新的菌毛结构。这些菌毛由一个18 kDa的菌毛蛋白亚基组成，其氨基末端与EDL933染色体上的ycbQ基因预测产物相同。序列比较分析表明，这些菌毛，这里称为YcbQ，属于毒力相关菌毛家族，由在几个动物和人类致病性大肠杆菌菌株中发现的F17，K99和G菌毛，以及铜绿假单胞菌的铜绿假单胞菌菌毛组成。通过对EDL933基因组序列的同源性分析，确定了与F17家族纤毛基因同源的4个基因(ycbQ、ycbR、ycbS、ycbT)，为结构基因ycbQ。总之，这项建议的目的是通过阐明EHEC O157：H7与培养的人上皮细胞黏附的机制(S)，促进对EHEC发病机制的认识。将结合分子生物学、细胞生物学、高功率电子显微镜超微结构分析、生化和抗原分析等多学科的方法，扩展我们目前对EHEC O157：H7与宿主靶细胞相互作用的认识。这项建议的结果将为EHEC疾病的诊断和食物来源和水库中O157：H7的检测提供重要意义。所获得的信息将对EHEC感染的预防和控制策略具有重要意义。这一建议的中心焦点在于以下具体目标：1)确定YcbQ菌毛生物发生所需的基因；2)确定YcbQ菌毛的作用；以及3)研究Ycb基因的转录表达。

DESCRIPTION (provided by applicant): Enterohemorrhagic E. coil (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 and purified a novel pilus structure produced by EHEC strain EDL933 and other O157:H7 strains. These pili are composed of an 18- kDa pilin subunit and its amino terminus shows identity to the predicted product of the ycbQ gene contained in the EDL933 chromosome. Sequence comparison analysis revealed that these pili, herein called YcbQ, belong to the virulence-associated pili family composed of F17, K99, and G pili found in several animal and human pathogenic E. coli strains, and CupA pili of Pseudomonas aeruginosa. Four genes (ycbQ, ycbR, ycbS, ycbT) with homology to F17 family piliation genes were identified by sequence homology in the genome of EDL933, being ycbQ the structural gene. 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 carded 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 diagnosis of EHEC disease and detection of O157:H7 in food sources and reservoirs. The information obtained will be important for prevention and control strategies of EHEC infections. The central focus of this proposal lies in the following specific aims: 1) To define the genes required for YcbQ pili biogenesis; 2) Define the role of YcbQ pili; and 3) Study transcriptional expression of ycb genes.