Two atypical enteropathogenic Escherichia coli strains induce the production of secreted and membrane-bound mucins to benefit their own growth at the apical surface of human mucin-secreting intestinal HT29-MTX cells

Abstract

In rabbit ligated ileal loops, two atypical enteropathogenic Escherichia coli (aEPEC) strains, 3991-1 and 0421-1, intimately associated with the cell membrane, forming the characteristic EPEC attachment and effacement lesion of the brush border, induced a mucous hypersecretion, whereas typical EPEC (tEPEC) strain E2348/69 did not. Using cultured human mucin-secreting intestinal HT29-MTX cells, we demonstrate that apically aEPEC infection is followed by increased production of secreted MUC2 and MUC5AC mucins and membrane-bound MUC3 and MUC4 mucins. The transcription of the MUC5AC and MUC4 genes was transiently upregulated after aEPEC infection. We provide evidence that the apically adhering aEPEC cells exploit the mucins' increased production since they grew in the presence of membrane-bound mucins, whereas tEPEC did not. The data described herein report a putative new virulence phenomenon in aEPEC.

FIG. 1.

aEPEC strains 3991-1 and 0421-1 promote an increase in fluid secretion in rabbit ligated ileal loops. Rabbit ligated ileal loops received LB broth or E. coli HB101, aEPEC 3991-1, aEPEC 0421-1, or tEPEC E2348/69 bacteria as described in Materials and Methods. The photograph in panel A shows that fluid secretion was increased in the presence of aEPEC 3991-1 and to a lesser extent in the presence of aEPEC 0421-1 bacteria, but not in the presence of HB101 and tEPEC E2348/69 bacteria. In panel B, excision of an ileal loop infected with aEPEC 3991-1 bacteria reveals the mucin nature of the increased fluid secretion. The photographs are representative of six rabbits examined.

FIG. 2.

Adhering aEPEC 3991-1 and 0421-1 bacteria formed large clusters of bacteria at the brush border of rabbit intestinal epithelium. In panel A, Giemsa coloration of aEPEC 3991-1- or 0421-1-infected rabbit intestinal epithelium showing the randomly distributed large aEPEC clusters. In panel B, transmission electron microscopy examination of aEPEC 3991-1- or 0421-1-infected rabbit intestinal epithelium showing the adhering bacteria intimately associated with the cell membrane and forming the typical EPEC attachment and effacement lesion of the brush border. In panel C, periodic acid-Schiff coloration of aEPEC 3991-1- or 0421-1-infected rabbit intestinal epithelium showing an increase of PAS staining relative to tEPEC infected. As a negative control, infection was conducted with E. coli HB101 bacteria.

FIG. 3.

Adhesion of aEPEC 3991-1 and 0421-1 strains and tEPEC strain E2348/69 to fully differentiated, mucus-secreting HT29-MTX cells. Cell monolayers were infected (10⁸ CFU/ml) and incubated at 37°C in a 10% CO₂-90% air atmosphere. After 3 h of infection, the cells were lysed with sterile water for the determination of cell-associated bacteria by a colony count assay. Each value shown is the mean ± SD from three experiments (three successive passages of cultured cells) in triplicate. *, P < 0.01 as compared with adhesion at 3 h postinfection for aEPEC 0421-1 and tEPEC E2348/69.

FIG. 4.

Production of MUC2 and MUC5AC secreted mucins and MUC3 and MUC4 membrane-bound mucins in mucin-secreting HT29-MTX cells infected or not with aEPEC 3991-1 or aEPEC 0421-1 or tEPEC E2348/69 strains. Indirect immunofluorescence labeling was monitored in nonpermeabilized cells with anti-MUC2 MAb, anti-MUC5AC MAb 1-13 M1, anti-MUC3 polyclonal antibody (pAb), or anti-MUC4 pAb and appropriate secondary FITC-conjugated antibodies. Micrographs are representative of two experiments (two successive passages of cultured cells).

FIG. 5.

Production of secreted MUC5AC mucin in control mucin-secreting HT29-MTX cells, and in cells infected with aEPEC 3991-1 or aEPEC 0421-1 or tEPEC E2348/69 strains. Culture media of control and infected cells were analyzed for determination of MUC5AC mucin, determined by immunoradiometric assay using the anti-MUC5AC 1-13M1 and PM7 MAbs. In panel A, MUC5AC mucin in control and cells with 3-h bacterial infection cells. In panel B, MUC5AC mucin in cells subjected for 3 h to LB broth (control) or the cell-free spent culture supernatant from an 18-h culture of L. monocytogenes strain EGD or the aEPEC 3991-1, aEPEC 0421-1, or tEPEC E2348/69 strains. L. monocytogenes strain EGD CFCS was used as a positive control for the stimulation of MUC5AC secretion in HT29-MTX by listeriolysin O toxin (47). Each value shown is the mean ± standard error of the mean from three experiments (three successive passages of cultured cells). Statistical analysis was performed with Student's t test. *, P < 0.01 for L. monocytogenes EGD-infected cells compared to control uninfected cells and aEPEC 3991-1 or aEPEC 0421-1-infected cells.

FIG. 6.

Determination of MUC2, MUC5AC, and MUC4 mucin mRNA expression in control mucin-secreting HT29-MTX cells and in cells infected with the aEPEC 3991-1, aEPEC 0421-1, or tEPEC E2348/69 strains. qRT-PCR was monitored as described in Materials and Methods. Noninfected HT29-MTX cells have a value of 1. Each value shown is the mean ± standard error of the mean from three experiments (three successive passages of cultured cells).

FIG. 7.

Association of aEPEC 3991-1 and 0421-1 bacterial cells with and internalization within mucin-secreting HT29-MTX cells and enterocyte-like HT-29 glc^−/+ cells. Cell monolayers were infected (10⁸ CFU/ml) and incubated for 3 h at 37°C in a 10% CO₂-90% air atmosphere. For the determination of cell-associated bacteria, infected cells were washed with sterile PBS and lysed with sterile water. To determine the internalized bacteria, the infected cells were washed with sterile PBS, incubated for 1 h in a medium containing 100 μg/ml of gentamicin to kill extracellular bacteria, and lysed with sterile water. Appropriate dilutions were plated on LB agar to determine the number of viable intracellular bacteria by bacterial colony counts. Each value shown is the mean ± SD of three experiments (three successive passages of cultured cells).

FIG. 8.

Growth of aEPEC 3991-1 and 0421-1 and tEPEC E2348/69 bacteria in the presence of secreted and/or membrane-bound mucins. Cell monolayers were infected (5 × 10⁷ CFU/ml) and incubated at 37°C in a 10% CO₂-90% air atmosphere. In panel A, after the secreted mucins had been eliminated by washing from infected HT29-MTX cells, an increased growth of adhering aEPEC 3991-1 and 0421-1 cells, but not of the tEPEC E2348/69 bacteria, develops. Panel B shows growth of aEPEC 3991-1, aEPEC 0421-1, and tEPEC E2348/69 bacteria (2 × 10⁷ CFU/ml) in DMEM or in the presence of DMEM culture medium of ATP-stimulated HT29-MTX cells containing secreted mucins. Panel C shows growth of aEPEC 3991-1 and 0421-1 cells and tEPEC E2348/69 bacteria that adhered to enterocyte-like Caco-2/TC7 cells in the presence or not of HT29-MTX secreted mucins. Each value shown is the mean ± SD of three experiments (three successive passages of cultured cells). *, P < 0.01 as compared to the adhesion 3 h postinfection plus washing.