Shiga toxin-producing Escherichia coli can impair T84 cell structure and function without inducing attaching/effacing lesions

Abstract

Enteropathogenic Escherichia coli (EPEC) intimately adhere to epithelial cells producing cytoskeletal rearrangement with typical attaching and effacing lesions and altered epithelial barrier and transport function. Since EPEC and Shiga toxin-producing E. coli (STEC) share similar genes in the "locus for enterocyte effacement" (LEE) thought to cause these changes, it has been assumed that STEC shares similar pathogenic mechanisms with EPEC. The aims of this study were to compare the effects of EPEC and STEC on bacterial-epithelial interactions and to examine changes in epithelial function. T84 monolayers were infected with STEC O157:H7 (wild strain EDL 933 or non-toxin-producing strain 85/170), EPEC (strain E2348/69), or HB101 (nonpathogenic) and studied at various times after infection. EPEC bound more avidly than EDL 933, but both strains exhibited greater binding than HB101. Attaching and effacing lesions and severe disruption to the actin cytoskeleton were observed in EPEC by 3 h postinfection but not in EDL 933 or HB101 at any time point. EPEC and EDL 933 increased monolayer permeability to [(3)H]mannitol 5- to 10-fold. In contrast to EPEC, EDL 933 completely abolished secretagogue-stimulated anion secretion as assessed under voltage clamp conditions in Ussing chambers. Several other STEC strains induced changes similar to those of EDL 933. In conclusion, STEC impairs epithelial barrier function and ion transport without causing major disruption to the actin cytoskeleton. Pathogenic factors other than products of LEE may be operant in STEC.

FIG. 1

Binding of HB101, EPEC, and STEC to T84 cells. Values are the means ± the standard error of the percentage of the inoculation dose that bound to the monolayers at 3, 6, and 18 h postinfection. For each group, n = 6. Significance values: ∗, P < 0.01 compared to HB101; ∗∗, P < 0.01 compared to EPEC.

FIG. 2

Immunofluorescent micrographs of actin cytoskeleton (A, C, E, and G) and microtubules (B, D, F, and H) at 18 h after infection. Actin cytoskeleton was stained with FITC-phalloidin and microtubules with anti-β-tubulin monoclonal antibody. Control monolayers (A and B) were compared with monolayers infected with BH101 (C and D), EPEC (E and F), and STEC (G and H). Arrows depict the sites of actin rearrangement under adherent bacteria. Bar, 10 μm.

FIG. 3

Electron micrographs of control (A) and HB101 (B)-, EPEC (C)-, and STEC (D)-infected T84 cells. Arrows in panel C depict A/E lesions of EPEC.

FIG. 4

Immunofluorescent micrographs of microtubules at 6 h after infection with STEC (A) and EPEC (B). Bar, 10 μm.

FIG. 5

Mucosal-to-serosal flux of [³H]mannitol (A) and IBMX-stimulated short-circuit current (ΔIsc) (B) in T84 cells infected for either 6 or 18 h. Values are the means ± the standard error (n = 8 to 16 monolayers in each group). Significance: ∗, P < 0.01 compared to control; and ∗∗, P < 0.001 compared to HB101 in panel A; ∗, P < 0.05 compared to control; ∗∗, P < 0.001 compared to control; and +, P < 0.001 compared to HB101 in panel B.