Study on molecular basis of species-specific adhesion of enteropathogenic Escherichia coli to host cells.

肠致病性大肠杆菌（EPEC）是人类腹泻的病原体。在体外粘附试验中，用培养的人上皮细胞再现了EPEC在肠粘膜上的局部粘附。我们发现EPEC粘附于小鼠来源的结肠上皮细胞系CMT-93的效率显著低于其粘附于人来源的肠细胞系，如肠-407或Caco-2。尽管EPEC确实粘附于一些来源于非人类物种的细胞系，但用福尔马林固定细胞以抑制一种或多种福尔马林敏感因子使我们能够观察到EPEC粘附的物种特异性差异。与这些结果相反，EPEC突变体，这是缺陷的，在形成菌毛皮利（BFP）的生产，有效地粘附到CMT-93细胞作为Caco-2细胞。此外，纯化的BfpA-His 6融合蛋白对Caco-2细胞的亲和力高于CMT-93细胞，并抑制EPEC粘附。这些结果表明BFP在细胞类型依赖性粘附中起重要作用。另一方面，我们确定了BFP在EPEC依从性中的作用。BFP是自聚集和小菌落形成所必需的。EPEC在上皮细胞上表达的BFP随着小集落的扩增而消失。通过与宿主细胞膜提取物接触诱导细菌分散和从EPEC聚集体释放BFP。因此，表达BFP的EPEC直接粘附于细胞表面，优先于粘附于细胞上预先形成的小菌落。这些结果表明，BFP介导EPEC的初始附着是通过与宿主细胞的直接相互作用，而不是将未附着的细菌募集到细胞上的小菌落。

Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhea in humans. Localized adherence of EPEC onto intestinal mucosa was reproduced in an in vitro adherence assay with cultured human epithelial cells. We found that the efficiency of EPEC adherence to a mouse-derived colonic epithelial cell line, CMT-93, was remarkably lower than its adherence to human-derived intestinal cell lines, such as Intestine-407, or Caco-2. Although EPEC did adhere to some cell lines derived non-human species, fixing the cells with formalin to inactivate one or more formalin-sensitive factors allowed us to observe species-specific differences in EPEC adherence. In contrast to these results, an EPEC mutant that is defective in bundle-forming pili (BFP) production adhered as efficiently to CMT-93 cells as to Caco-2 cells. Furthermore, a purified BfpA-His6 fusion protein showed higher affinity for Caco-2 cells than for CMT-93 cells, and inhibited EPEC adherence. These results indicated that BFP plays an important role in the cell-type-dependent adherence. On the other hand, we determined the role of BFP in EPEC adherence. BFP is necessary for autoaggregation and formation of microcolony. BFP expressed by EPEC on epithelial cells was shown to disappear in accordance with expansion of microcolony. Bacterial dispersal and release of BFP from EPEC aggregates were induced by contact with host cellular membrane extract. Consequently, BFP-expressing EPEC adhered directly to the surface of cells, in preference to attaching to pre-formed microcolonies on the cells. These results suggested that BFP mediate initial attachment of EPEC through direct interaction with host cell rather recruitment of unattached bacteria to microcolony on the cell.