Cleary Jennifer, Lai Li-Ching, Shaw Robert K, Straatman-Iwanowska Anna, Donnenberg Michael S, Frankel Gad, Knutton Stuart
Institute of Child Health, University of Birmingham, Birmingham, UK.
Division of Infectious Diseases, University of Maryland, Baltimore, MD, USA.
Microbiology (Reading). 2004 Mar;150(Pt 3):527-538. doi: 10.1099/mic.0.26740-0.
Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic 'attaching and effacing' (A/E) lesions on small intestinal enterocytes. EPEC adhesins that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and intimin. BFP are involved in bacteria-bacteria interaction and microcolony formation but their role in cell adhesion remains unclear; EspA filaments are components of the EPEC type III secretion system but since they interact directly with host cells they may also function as adhesins; intimin is the well characterized intimate EPEC adhesin which binds the translocated intimin receptor, Tir. However, other uncharacterized host cell receptors have been implicated in intimin-mediated adhesion. In this study, the role of BFP, EspA filaments and intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in bfpA, espA and eae (intimin gene) to differentiated human intestinal Caco-2 cells. E2348/69 (bfpA(+) espA(+) eae(+)) adhered rapidly (<10 min) to the brush border of Caco-2 cells and subsequently produced microcolonies and typical A/E lesions. Non-intimate brush border adhesion of double mutant strain UMD880 (bfpA(+) espA(-) eae(-)) also occurred rapidly, whereas adhesion of strain UMD886 (bfpA(-) espA(+) eae(-)) occurred later in the infection (>1 h) and with much lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively. Strain UMD883 (bfpA(-) espA(-) eae(+)), which is unable to translocate Tir, was non-adherent although this strain was able to form intimate attachment and A/E lesions when co-cultured with strain CVD206 (bfpA(+) espA(+) eae(-)) which supplied Tir to the membrane. Single mutant strains CVD206 (bfpA(+) espA(+) eae(-)) and UMD872 (bfpA(+) espA(-) eae(+)) showed adherence characteristics of strain UMD880 (bfpA(+) espA(-) eae(-)), whilst triple mutant strain UMD888 (bfpA(-) espA(-) eae(-)) was totally non-adherent. These results support an adhesive role for BFP and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as initial attachment factors in atypical EPEC which lacks BFP. The study found no evidence for an independent host cell intimin receptor or for other adhesive factors able to support bacterial adherence.
肠致病性大肠杆菌（EPEC）是一种重要的引起小儿腹泻的病原体，它利用多种黏附素定殖于小肠，并在小肠肠上皮细胞上产生特征性的“紧密黏附并消除”（A/E）损伤。与A/E黏附及肠道定殖相关的EPEC黏附素包括束状菌毛（BFP）、EspA细丝和紧密黏附素。BFP参与细菌-细菌相互作用和微菌落形成，但其在细胞黏附中的作用仍不清楚；EspA细丝是EPECⅢ型分泌系统的组成部分，但由于它们直接与宿主细胞相互作用，因此也可能起到黏附素的作用；紧密黏附素是特征明确的EPEC紧密黏附素，它与转位紧密黏附素受体Tir结合。然而，其他未明确的宿主细胞受体也与紧密黏附素介导的黏附有关。在本研究中，通过观察野生型EPEC菌株E2348/69以及bfpA、espA和eae（紧密黏附素基因）的一组同基因单突变、双突变和三突变体与分化的人肠道Caco-2细胞的黏附情况，评估了BFP、EspA细丝和紧密黏附素在EPEC黏附于肠道刷状缘细胞中的作用。E2348/69（bfpA(+) espA(+) eae(+)）迅速（<10分钟）黏附于Caco-2细胞的刷状缘，随后形成微菌落和典型的A/E损伤。双突变菌株UMD880（bfpA(+) espA(-) eae(-)）的非紧密刷状缘黏附也迅速发生，而菌株UMD886（bfpA(-) espA(+) eae(-)）在感染后期（>1小时）发生黏附，且效率低得多；共聚焦显微镜分别显示了BFP和EspA介导的黏附。无法转位Tir的菌株UMD883（bfpA(-) espA(-) eae(+)）不黏附，尽管该菌株与提供Tir至细胞膜的菌株CVD206（bfpA(+) espA(+) eae(-)）共培养时能够形成紧密附着和A/E损伤。单突变菌株CVD206（bfpA(+) espA(+) eae(-)）和UMD872（bfpA(+) espA(-) eae(+)）表现出菌株UMD880（bfpA(+) espA(-) eae(-)）的黏附特征，而三突变菌株UMD888（bfpA(-) espA(-) eae(-)）则完全不黏附。这些结果支持BFP和EspA在初始刷状缘细胞黏附中具有黏附作用，并且在同时表达BFP和EspA细丝的典型EPEC中，表明BFP起主要作用；然而，EspA细丝可作为缺乏BFP的非典型EPEC中的初始黏附因子。该研究未发现独立的宿主细胞紧密黏附素受体或其他能够支持细菌黏附的黏附因子的证据。
