Fig. 2.

Recombinant RrgA and RrgC proteins bind to human respiratory epithelial cells. A–C. Recombinant RrgA binds directly to A549 respiratory epithelial cells. Cells were incubated with 100 μg ml⁻¹ of purified RrgA (A1 and A2, ‘+RrgA’) or GFP (B1 and B2, ‘+GFP’) in DMEM culture medium, or medium alone (A3–4, ‘-RrgA’; B3–4, ‘-GFP’) for 2 h at 4°C. Cells were fixed and stained with anti-RrgA and anti-GFP antibodies (A1–4, ‘αRrgA’; B1–4, ‘αGFP’) and phalloidin, which served as a control to demonstrate the presence of cells (A2, A4, B2 and B4). Imaging was performed with a confocal microscope. Scale bar is 20 μm. C. RrgA and RrgC bind to A549 cells in a dose-dependent manner. A549 cells were mixed in suspension with either medium alone (0 μg ml⁻¹) or three concentrations (5, 50 and 100 μg ml⁻¹) of pilus subunits RrgA (squares with solid black lines), RrgB (triangles with dashed grey lines), RrgC (upside-down triangles with dashed black lines), or GFP protein (diamond with solid grey lines), incubated for 2 h at 4°C, stained with antisera specific to each protein, and detected with Alexa Fluor 488-conjugated secondaries. Cells were analysed with a FACS-Calibur flow cytometer, and the net mean fluorescence intensity for each population was calculated from three independent experiments. Significant differences were detected by repeated-measure anova (P< 0.0001), and both RrgA and RrgC binding was significantly different from RrgB and GFP binding at 50 and 100 μg ml⁻¹ by post hoc Bonferroni analysis (*P < 0.001). D–G. Pre-incubation of A549 cells with purified RrgA protein inhibits pilus-mediated adherence. A549 cells were pre-incubated with media alone (D), media containing 100 μg ml⁻¹ of RrgA (E), or 100 μg ml⁻¹ of GFP (F). After pre-incubation, A549 monolayers were infected with S. pneumoniae strain T4. Cells were stained with phalloidin (red) anti-S. pneumoniae capsule antibody (green), and imaged with a confocal microscope. RrgA pre-incubation inhibits the adherence of strain T4 to the A549 cells (E versus D), while the negative control GFP protein does not (F versus D). Scale bar is 20 μm. Adherent bacteria were counted, and the number of bacteria adherent to 100 A549 cells is shown in G (n = 6 fields, *P= 0.0002, **P= 0.8).