Figure 7.

Human rMUC1-ED inhibits flagellin binding and Pa adhesion to human airway ECs and Pa motility. A, PAK was incubated with 25 μm of human rMUC1-ED for 1 h at 4 °C. The bacteria were incubated with mouse anti-MUC1-ED Ab (panel i) or nonimmune mouse IgG (panel ii), each at 1:5000 dilution, for 1 h at 4 °C, followed by gold-labeled goat anti-mouse IgG secondary Ab at 1:10,000 dilution for 1 h at 4 °C, and examined by transmission immunoelectron microscopy. Each section was photographed at 11,000× or 44,000× (insert). Arrowheads indicate immunogold labeling. Scale bar, 100 nm. B and C, Human rMUC1-ED at the indicated concentrations or the PBS vehicle control were incubated with (B) Alexa Fluor 594–labeled Pa flagellin (n = 3) or (C) WT PAK or the flagellin-deficient PAK/fliC⁻ mutant (n = 3) and assayed for (B) flagellin binding or (C) Pa adhesion to Ad-NEU1–infected A549 cells. D, the indicated concentrations of human rMUC1-ED or the PBS vehicle control were incubated with WT PAK or the PAK/fliC⁻ mutant, and the bacteria were stab-inoculated into 0.3% agar plates and incubated overnight. Pa colony diameter was measured as an indicator of bacterial motility (n = 3). Error bars represent mean ± S.E. flagellin binding (B), Pa adhesion (C), or Pa motility (D). **, significantly decreased flagellin binding or Pa adhesion or motility versus the PBS control at p < 0.05. The results are representative of three independent experiments.