Figure 4: Lpf are needed for active translocation of EHEC strain EDL933 across M-cell monolayers and in vivo interactions with murine Peyer’s patches.

Caco-2 cl1 and M-cells were infected with 10⁷ EHEC bacteria (wild-type EHEC O157:H7 strain EDL933, lpf isogenic mutants, trans-complemented strains and O6:H10 strain NV110) per Transwell^TM. The number of translocated bacteria across Caco-2 cl1 (A) and M-cells (B) was determined at 2, 4 and 6 h post-infection by plating on selective agar plates. Results are expressed as mean CFU/ml ± standard deviation (n = 3). Translocation through Caco-2 cl1 or M-cells significantly different between wild-type strain EDL933 and isogenic mutants or O6:H10 NV110 at p < 0.001 (α). Translocation through Caco-2 cl1 or M-cells significantly different between isogenic mutants and trans-complemented strains at p < 0.001 (β). (C and D) Mice ileal loops (n = 10) were inoculated with a mixed inoculum containing equivalent number (5∙10⁸ CFU/ml) of EHEC O157:H7 EDL933 wild-type strain and either lpf isogenic mutants, trans-complemented mutants or the O6:H10 strain NV110. Bacterial interactions with ileal mucosa (C) or Peyer’s patches (D) were determined by competitive index (CI) analysis relative to the O157:H7 wild-type strain. Significant differences between CI obtained with isogenic mutants and the trans-complemented strains at p < 0.05 (*), p < 0.001 (**) or p < 0.001 (***). CI of O6:H10 significantly different from 1 at p < 0.001 (**). (E) Macroscopic view of hemorrhagic (left) or non-hemorrhagic (right) Peyer’s patches. (F) The percentages of hemorrhagic Peyer’s patches were determined in mice ileal loops infected with wild-type EHEC strain EDL933 (N = 32) or ΔlpfA_OI-141/ΔlpfA_OI-154 isogenic mutant (N = 26). Percentages obtained with ΔlpfA_OI-141/ΔlpfA_OI-154mutant significantly different from that of wild-type strain at p < 0.001 (***).