Invasive ability of an Escherichia coli strain isolated from the ileal mucosa of a patient with Crohn's disease

Abstract

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.

FIG. 1

PCR amplification products analysis. (A) One-kilobase DNA ladder (Life Technologies). Amplication products were generated by using primers for tia-specific sequence of ETEC strain H10407 (B), eae-specific sequence of EPEC (C), and ipaC-specific sequence of S. flexneri SC301 (D). DNA to be amplified was released by boiling from ETEC strain H10407 (B, lane 1), EPEC strain E2348/69 (C, lane 1), EIEC strain E12860/0 (D, lane 1), strain LF82 (lane 2), and S. flexneri SC301 (D, lane 3).

FIG. 2

Colony blot hybridizations of intragenic tia (A), eae (B), and ipaC (C) probes corresponding to radiolabeled PCR-amplified products generated with DNA templates from ETEC strain H10407, EPEC strain E2348/69, and S. flexneri SC301, respectively. (D) Colony blot hybridization of rrnB7 probe. Blots: 1, strain LF82; 2, EIEC strain 12860/0; 3, DAEC strain C1845; 4, EAggEC strain 17-2; 5, EPEC strain E2348/69; 6, ETEC strain H10407; 7, EHEC strain EDL933; 8, E. coli K-12 C600.

FIG. 3

Time course of bacterial entry. HEp-2 cells were infected at an MOI of 10 for various infection periods and treated with gentamicin for 1 h. Percent invasion is the mean percentage of the inoculum surviving gentamicin treatment. (A) ■, strain LF82; ▴, EPEC strain E2348/69; □, ETEC strain H10407. (B) ■, strain LF82; ○, EIEC strain E12860/0. Each point is the mean of at least three separate experiments.

FIG. 4

Kinetics of intracellular multiplication. HEp-2 cell monolayers were infected for 3 h, and the number of intracellular bacteria was determined after different times of gentamicin treatment. Results are expressed as the number of intracellular bacteria relative to that obtained at 1 h of gentamicin treatment, taken as 100%. (A) ■, strain LF82; ▴, EPEC strain E2348/69. (B) ■, strain LF82; ○, EIEC strain E12860/0. Each point is the mean of at least three separate experiments.

FIG. 5

Effects of eukaryotic cell inhibitors on association with (A and B) and invasion of (C and D) HEp-2 cells. HEp-2 cell monolayers were pretreated with cytochalasin D or colchicine for 30 min before the assay. Infections were performed for 3 h in the presence of the inhibitors. Cell-associated bacteria were quantified after the 3-h infection period. Invasion was determined after gentamicin treatment for an additional hour. Results are expressed as cell-associated or intracellular bacteria relative to those obtained without inhibitor, taken as 100%. ■, strain LF82; , EIEC strain E12860/0; ░⃞, EPEC strain E2348/69. Each value is the mean of at least three separate experiments.

FIG. 6

Transmission electron micrographs of HEp-2 cells infected with strain LF82. (A) Cross section of the cell monolayer showing numerous intracellular bacteria after a 5-h infection period. (B) Micrograph showing membrane ruffling upon contact with the bacteria after a 3-h infection period. Bacteria are engulfed by elongated microvilli from the infected epithelial cells. (C) High magnification showing a partially lysed vacuolar membrane containing bacteria, indicating the ability of strain LF82 to escape from the endocytic vacuoles. Magnifications: A, ×6,200; B, ×21,600; C, ×28,800.