FIG. 9.

The length of the LPS core determines OmpX-mediated internalization and adherence. (A) E. coli K-12 D21 (rough LPS) strain and its three isogenic derivatives D21e7 (rough LPS), D21f1 (deep rough LPS), and D21f2 (deep rough LPS) with or without pOmpX were grown to mid-exponential phase at 37°C and coincubated for 1 h with Hep-2 monolayers at 37°C (MOI = 20 to 50). Hep-2 host cells were disrupted and plated on LB agar or incubated with gentamicin for 1.5 h to kill extracellular bacteria before plating. Open bars represent the number of cell-associated bacteria (extracellular and intracellular), and filled bars represent the number of intracellular bacteria. Results are means ± SEM from assays performed in duplicate on two separate days (n = 8). The asterisk (*) indicates differences (ANOVA, P < 0.0001) between the strain expressing OmpX (pOmpX) and cell (D21, D21e7, D21f1, or D21f2) and plasmid (pTA) control strains. (B) Field emission scanning microscopy imaging of OmpX-expressing E. coli associated with Hep-2 cells. Bacteria and eukaryotic cells were grown as described above and cocultured for 15 min at 37°C. After extensive washing, the cells were fixed, treated with tannic acid, OsO₄, dehydrated, and gold coated. The samples were analyzed with Quanta 200F (Field Emission Instruments) at a magnification of ×16,000. The bar represents 5 μm; white arrows indicate bacteria associated with Hep-2 cells.