Invasion of epithelial cells by locus of enterocyte effacement-negative enterohemorrhagic Escherichia coli

Abstract

The majority of enterohemorrhagic Escherichia coli (EHEC) strains associated with severe disease carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the ability to induce attaching and effacing lesions on the host intestinal mucosa. While LEE is essential for colonization of the host in these pathogens, strains of EHEC that do not carry LEE are regularly isolated from patients with severe disease, although little is known about the way these organisms interact with the host epithelium. In this study, we compared the adherence properties of clinical isolates of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. Transmission electron microscopy revealed that LEE-negative EHEC O113:H21 was internalized by Chinese hamster ovary (CHO-K1) epithelial cells and that intracellular bacteria were located within a membrane-bound vacuole. In contrast, EHEC O157:H7 remained extracellular and intimately attached to the epithelial cell surface. Quantitative gentamicin protection assays confirmed that EHEC O113:H21 was invasive and also showed that several other serogroups of LEE-negative EHEC were internalized by CHO-K1 cells. Invasion by EHEC O113:H21 was significantly reduced in the presence of the cytoskeletal inhibitors cytochalasin D and colchicine and the pan-Rho GTPase inhibitor compactin, whereas the tyrosine kinase inhibitor genistein had no significant impact on bacterial invasion. In addition, we found that EHEC O113:H21 was invasive for the human colonic cell lines HCT-8 and Caco-2. Overall these studies suggest that isolates of LEE-negative EHEC may employ a mechanism of host cell invasion to colonize the intestinal mucosa.

FIG. 1.

Transmission electron microscopy of CHO-K1 cells infected with EHEC O113:H21 EH41 and EHEC O157:H7 EDL933. A. EH41. Magnification, ×5,000. Arrowheads indicate vacuolar membrane. B. EDL933. Magnification, ×5,000. C. EH41. Magnification, ×3,000. D. EDL933. Magnification, ×3,000.

FIG. 2.

Invasion of CHO-K1 epithelial cells by clinical isolates of LEE-negative EHEC. Results are expressed as the percentage of cell-associated bacteria that resisted killing by gentamicin and are the means ± standard deviations from at least three independent experiments in duplicate wells. 1) E. coli HB101; 2) E. coli DH5α; 3) EDL933; 4) 85-170; 5) E2348/69; 6) EI6/84; 7) EI223/83; 8) EH5; 9) EH42; 10) EH43; 11) EH69; 12) EH52; 13) EH53; 14) EH71; 15) EH41; 16) EH41c. *, significantly more than EDL933 (P < 0.05 by an unpaired, two-tailed t test).

FIG. 3.

Immunofluorescence microscopy of CHO-K1 cells infected with EHEC O113:H21 EH41 for 5 min, 15 min, and 30 min. Bacteria were stained with antibodies raised to O113:H21 surface components before and after permeabilization of CHO-K1 cells with Triton X-100. Intracellular bacteria appear green, and extracellular bacteria appear orange/yellow. The arrow indicates bacterial cells staining partly green and partly orange/yellow. The host cell cytoskeleton was stained with phalloidin-TRITC.