Outer membrane protein A of Escherichia coli O157:H7 stimulates dendritic cell activation

Abstract

Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.

FIG. 1.

Production of cytokines by OmpA-stimulated DCs. BM-DCs of (A through D) C3H/HeJ or (E and F) C57BL/6 mice were used at day 8. (A through D) DCs were left untreated (control) or stimulated with indicated concentrations (μg/ml) of purified OmpA or LPS. (E and F) Alternatively, purified OmpA or LPS was pretreated with (+) or without (−) polymyxin B (10 μg/ml) for 10 min before addition to DC cultures. Cell-free culture supernatants were collected at 24 h after stimulation for the measurement of (A) IL-1α, (B) IL-1β, (C and E) IL-10, and (D and F) IL-12p70 (pg/ml) by specific ELISA. Results are shown as means ± standard deviations for each group and are representative of (A to D) three or (E and F) two independent experiments. Cells stimulated with 0.04 μg/ml of LPS produced no cytokines or background levels of cytokines. *, P < 0.05; **, P < 0.01.

FIG. 2.

Expression of OmpA in EHEC O157:H7 wild-type and isogenic strains. (A) Wild-type EHEC O157:H7 strain 86-24 (lane 1), its isogenic ompA mutant strain (AGT601S) (lane 2), and the complemented strain AGT601S(pOmpA) (lane 3) were grown in LB media and fractionated on a 12% SDS-PAGE gel. Proteins transferred to a membrane were probed with anti (α)-OmpA PAb. The arrow indicates the OmpA protein. M, molecular mass markers (in kDa). (B) The duplicate SDS-PAGE gel indicates comparable loading of proteins in each lane. Note that while protein loads were generally comparable in all three samples, the AGT601S(pOmpA) strain had an increased expression level of an ∼24-kDa band, which is likely to be the product of the plasmid-encoded chloramphenicol resistance gene.

FIG. 3.

Production of cytokines by EHEC-stimulated DCs. BM-DCs of C3H/HeJ mice were cocultured with EHEC O157:H7 strain 86-24, AGT601S, AGT601S(pOmpA), or AGT601S (pACYC184) or E. coli K-12 strain MG1655 at indicated bacterium-to-cell ratios. Culture supernatants were collected at 24 h postinfection for the measurement of (A) IL-1α, (B) IL-1β, (C) IL-10, and (D) IL-12p70 (pg/ml) by specific ELISA. Results are shown as means ± standard deviations for each group and are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

FIG. 4.

Effect of anti-OmpA Abs on EHEC-stimulated DCs. BM-DCs of C3H/HeJ mice were used at day 8 and were stimulated with EHEC complemented strain AGT601S(pOmpA) at a bacterium-to-cell ratio of 1,000:1. Prior to the infection, the EHEC strain was incubated with indicated dilutions (vol/vol; Ab/bacterial suspension) of mouse anti-OmpA MAb or rabbit anti-OmpA PAb. An irrelevant mouse isotype Ab was used as a control. Culture supernatants were collected at 24 h postinfection for the measurement of (A) IL-1α, (B) IL-1β, and (C) IL-10 (ng/ml) by ELISA. Results are shown as means ± standard deviations for each group and are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

FIG. 5.

Ab-mediated enhancement in cytokine production requires the presence of bacteria. BM-DCs of C3H/HeJ mice were stimulated with the complemented strain AGT601S(pOmpA) at a bacterium-to-cell ratio of 1,000:1, using bacteria preincubated with indicated dilutions (vol/vol; Ab/bacterial suspension) of anti-OmpA MAb. DCs incubated with anti-OmpA MAb alone were used as controls. Culture supernatants were collected at 24 h postinfection for the measurement of (A) IL-1α, (B) IL-1β, and (C) IL-10 (pg/ml) by ELISA. Results are shown as means ± standard deviations for each group and are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

FIG. 6.

Adhesion of EHEC O157:H7 to and migration of human DCs across polarized Caco-2 cells. Caco-2 cells were infected with EHEC strain 86-24, its isogenic ompA mutant strain (AGT601S), or the complemented strain AGT601S(pOmpA) (at a bacterium-to-cell ratio of 2:1) for 1 h prior to the addition of human blood-derived DCs. The incubation was extended for an additional 4 h before samples were collected to determine (A) adhesion of bacteria to the apical surface of polarized Caco-2 cells [CFU/ml (10⁶)] and (B) the percentage of DCs migrated across polarized Caco-2 cells. Results are shown as means ± standard deviations for each group and are representative of two independent experiments. *, P < 0.05; **, P < 0.01.