Fig. 3

Inflammatory response-induced nitric oxide enhances EHEC’s colony-forming capacity. Caco-2 cells were exposed to a cytokine mix (IL-8, TNF-γ, IL-22) for 24 h. Culture supernatants were collected by centrifugation and filtration to remove cells and debris. EHEC O157 Sakai wild-type (WT) or the narGHJI mutant (ΔnarGHJI) were grown in the supernatant for 3 h under anaerobic conditions. HeLa cells were infected with EHEC for 5 h, including 3.5 h post-washing. Microcolonies were visualized by staining with Giemsa. a. Effect of culture supernatant of cells in inflammation. The wild-type EHEC strain was infected into HeLa cells after growth in the supernatant. b. Requirement of iNOS activity for enhancement of the microcolony-forming capacity. Caco-2 cells were exposed to a cytokine mix with or without aminoguanidine hydrochloride (AG), and culture supernatants were used for pre-culture of EHEC. Microcolonies on HeLa cells were determined as in A. c. Activation of nitrate respiration was necessary for the enhancement. EHEC O157 Sakai wild-type (WT) or the narGHJI mutant (ΔnarGHJI) were precultured in the supernatants and infected HeLa cells. The average of three independent experiments along with standard error is shown. *: p < 0.05, ns: not significant