Boudeau J, Glasser A L, Masseret E, Joly B, Darfeuille-Michaud A
Pathogénie Bactérienne Intestinale, Laboratoire de Bactériologie, Faculté de Pharmacie, 63001 Clermont-Ferrand, France.
Infect Immun. 1999 Sep;67(9):4499-509. doi: 10.1128/IAI.67.9.4499-4509.1999.
Crohn's disease (CD) is an inflammatory bowel disease in which Escherichia coli strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by E. coli strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic E. coli strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC) E. coli strains. Gentamicin protection assays showed that E. coli LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (P > 0.5) but significantly higher than that of ETEC (P < 0.03), EHEC (P < 0. 005), EAggEC (P < 0.004) and DAEC (P < 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- or Shigella-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive E. coli strain and suggest the existence of a new potentially pathogenic group of E. coli, which we propose be designated adherent-invasive E. coli.
克罗恩病（CD）是一种炎症性肠病，人们怀疑大肠杆菌菌株与该病有关。我们之前证明，CD的回肠病变部位定殖有能黏附于肠道Caco-2细胞的大肠杆菌菌株，但这些菌株缺乏目前在涉及胃肠道感染的致病性大肠杆菌菌株中所描述的毒力基因。在本研究中，我们比较了从一名CD患者的回肠活检样本中分离出的其中一株菌株LF82与参考肠侵袭性（EIEC）、肠致病性（EPEC）、产肠毒素性（ETEC）、肠集聚性（EAggEC）、肠出血性（EHEC）及弥漫黏附性（DAEC）大肠杆菌菌株的侵袭能力。庆大霉素保护试验表明，大肠杆菌LF82能够有效地侵袭HEp-2细胞。其侵袭水平与EIEC和EPEC菌株相比无显著差异（P>0.5），但显著高于ETEC（P<0.03）、EHEC（P<0.005）、EAggEC（P<0.004）和DAEC（P<0.02）菌株。菌株LF82还表现出有效侵袭肠道上皮培养的Caco-2、Intestine-407和HCT-8细胞的能力。对感染的HEp-2细胞进行电子显微镜检查发现，有大量细胞内细菌存在于液泡中或游离于宿主细胞质中。此外，菌株LF82与上皮细胞的相互作用伴随着从宿主细胞膜伸出并吞噬细菌的微绒毛延伸的延长。这种内化机制与沙门氏菌或志贺氏菌诱导的巨吞饮作用非常相似。细胞松弛素D和秋水仙碱的使用表明，HEp-2细胞对菌株LF82的摄取是由肌动蛋白微丝依赖性机制和微管参与介导的。此外，菌株LF82在HEp-2和Intestine-407细胞中存活至少24小时，并在HEp-2细胞内有效复制。PCR和杂交实验未发现存在任何编码参与细菌侵袭的EIEC、EPEC或ETEC蛋白的遗传决定因素。因此，这些发现表明，定殖于一名CD患者回肠黏膜的LF82是一种真正具有侵袭性的大肠杆菌菌株，并提示存在一种新的潜在致病性大肠杆菌群，我们建议将其命名为黏附侵袭性大肠杆菌。
