Fig 1. Mucosal carbohydrate-mediated binding of S. pneumoniae to mucus particles.

(A-D) Adherence of Spn Type 4 (TIGR4) to murine nasal lavages (mNL) was analyzed in a solid-phase assay. Bacteria (2x 10⁴ CFU/100 μl DMEM) were incubated with 100μL of undiluted, immobilized, pooled mNL in presence of 0.1% BSA for 2hr at 30°C. After 19 washes, adherent bacteria were determined by resuspending with 0.001% Triton X-100 followed by plating on TS agar plates supplemented with 200 μg/ml streptomycin. (A) Prior to immobilization, mNL was sonicated (Amplitude 8μM) for increasing amounts of time followed by blocking with 0.1% BSA and incubation with Spn (B) Filtering of mNL’s with a 0.45uM filter followed by immobilization, blocking with 0.1% BSA and incubation with Spn (C) Treatment of immobilized mNL with 100mM NaIO₄ in 50mM sodium acetate buffer for 30 min at 4°C in the dark followed by blocking with 0.1% BSA and incubation with Spn (D) Treatment of immobilized mNL with 50μg/mL trypsin for 30 min at 37°C followed by the blocking with 0.1% BSA and incubation with Spn. (E-G) Wild-type Spn were incubated with mNL or hNF for 2hr at 37°C and 5% CO₂. Scale bar represents 10μm. (E) Type 4 (TIGR4) incubated with mNL; (i-iv) Represents Type 4 (TIGR4) interacting with alcian blue staining material (open-arrows) (v) Type 4 (TIGR4) as diplococci or short chains not interacting with alcian blue staining material, bacteria shown with solid arrows (F) Spn Type 23F incubated with human nasal fluid (hNF). Mucus (blue) was stained with alcian blue and bacteria (green) were detected using rabbit anti-capsule antibody and secondary FITC-coupled goat anti-rabbit IgG. Spn were visualized by microscopy on an Axiovert 40 CFL microscope equipped with an Axiocam IC digital camera at 100x. (G) Spn Type 23F incubated with human nasal fluid (hNF). Mucus (green) was stained with a Muc5AC monoclonal antibody and bacteria (orange) were detected using rabbit anti-capsule antibody. Spn were visualized by microscopy on a LM- Zeiss AxioObserver at 63x. Experiments were performed in duplicates and mean values of three independent experiments are shown with error bars corresponding to S.D. *,p<0.05; **,p<0.01;***, p<0.001 by Kruskal-Wallis test, followed by Dunn’s multiple comparison test for multiple comparison (A) or an unpaired-T test (B,C, D).