Figure 3

Flagellin-expressing Pa induces MUC1-ED shedding in vitro. (A) Bacterial motility assays were used to verify flagellin expression by S. maltophilia, L. pneumophila, and S. enterica serovar Typhimurium. WT-PAK and the PAK/fliC¯ flagellin-deficient strain were used as positive and negative controls, respectively. Bars represent mean ± S.E. colony diameters (mm) as a functional assay for motility (n = 5). *, increased motility compared with the PAK/fliC¯ strain at p < 0.05. (B) To establish the time profile for MUC1-ED shedding, A549 cells (2.0 × 10⁵ cells/well) cultured in 24-well plates were incubated for increasing times with fixed inocula of either WT-PAK or the PAK/fiC¯ mutant (1.0 × 10⁷ CFUs/well). At each time point, MUC1-ED levels in cell culture supernatants were measured by ELISA and normalized to total A549 cell protein. (C) A549 cells (2.0 × 10⁵ cells/well) in 24-well plates were incubated for 24 h at 37 °C with 1.0 × 10⁷ CFUs/well of the indicated bacterial strains, 100 ng/well of Pa LPS, 10 µg/well of Pam₃Cys-Ser-(Lys)₄, 10 µg/well of CpG ODN 1826, 10 ng/well of Pa or STm flagellins, 10 ng/well of Pa rFlaA or rFla flagellins, or the PBS vehicle control. MUC1-ED levels in cell culture supernatants were measured by ELISA and normalized to total A549 cell protein. (D) A549 cells (2.0 × 10⁵ cells/well) cultured in 24-well plates were incubated for 24 h at 37 °C with increasing CFUs/well of WT-PAK or the PAK/fliC¯ flagellin-deficient strain. MUC1-ED levels in cell culture supernatants were measured by ELISA and normalized to total cell protein. (B–D) Bars and data points represent mean ± S.E. values (n = 3 or 5). (A, C) Open circles represent individual values. *, increased MUC1-ED levels compared with (A, B, D) PAK/fliC¯ or (C) the PBS control at p < 0.05. **, decreased MUC1-ED levels provoked by the PAK/fliC¯ flagellin-deficient strain compared with WT-PAK at p < 0.05. (E) WT-PAK (lanes 1, 3, 5) or the PAK/fliC¯ mutant strain (lanes 2, 4, 6) were co-cultured for 24 h with A549 cells, the supernatants collected, and centrifuged at 5,000 xg for 10 min to collect the bacteria. The bacteria were lysed and equal protein aliquots (10 µg) of the lysates processed for MUC1-ED immunoblotting to detect Pa:MUC1-ED complexes. Molecular weights in kDa are indicated on the left. The arrow indicates the > 250 kDa MUC1-ED band of interest. IB, immunoblot. The full-length immunoblot is shown. The results are representative of 2 or 3 independent experiments.