The role of adenylate cyclase toxin in <i>Bp</i> biofilm formation on HBE cells.

(A) Biofilm formation after 96 h was evaluated by GFP-labeled bacteria in microtiter plates by crystal violet method. Bars indicate the mean and standard error of the mean of two independent experiments performed by triplicate. Significant differences were calculated by using one-way ANOVA and Bonferroni post hoc. ***, p t-test. n.s., not significant. (C-D) HBE cells infected with GFP-expressing WT (C) or ΔcyaA (D) strains for 48 h were fixed and imaged using an Olympus FV3000 confocal microscope (20X objective). Z-stacks were acquired at 1-μm intervals. IMARIS software was used to produce a shading picture of the biofilms. (E) Representative live cell images of GFP-labeled bacteria on apical surface of HBE cells after 48 h (10X objective). (F) Scanning electron microscopy of bacterial strains 24 h after infection of HBE cells. For C-F, images are representative of at least two biological replicates.