0.22 μm filter unit

Capsular polysaccharides were isolated as described, with some modifications [5 (link), 18 (link)]. Briefly, cells were pelleted from cultures at 3,450 x g for 15 min at 4°C. Five mg of glass beads (4 mm, Fisher) per g of cells were added and the mixture was gently shaken by vortex for 1 min. A volume of 50 ml of distilled water was added per g of disrupted cells and centrifuged at 8000 x g for 10 min at 4°C. The supernatant was recovered, clarified in a 0.22 μm filter unit (Millipore) and lyophilized. To separate the capsular arabinomannan (AM) from the rest of capsular polysaccharides, the capsule residue was resuspended in 4 ml of distilled water and subjected to a chloroform:methanol:water extraction (1:1:0.9). The upper phase was recovered and incubated in a rotavapor at 40°C overnight. Proteinase K (Sigma) was added at 10 mg/ml in a 50 mM Tris-HCl pH 7.5, 10 mM CaCl₂ buffer and incubated overnight at 37°C. The deproteinated solution was dialyzed for 3 d at 4°C in distilled water, lyophilized and chromatographed on a column (90 cm x 1.8 cm) of Bio-Gel P-10 (Bio-Rad) using 0.1 M NaCl in 0.1% acetic acid. Collected fractions of 4 ml were assayed for carbohydrate content by the phenol-sulfuric acid assay. Pooled fractions were dialyzed in water and lyophilized. The concentration of protein was determined on each isolation step by Bradford.

Tannic acid was obtained from MP Biomedicals LLC, Solon, OH, USA and Post Apple Scientific Inc., North East, PA, USA. Tannic acid was dissolved in deionized water and sterilized using a 0.22 μM filter unit (Millipore) and 5 mM stock solutions were prepared. Heparin and gallic acid were purchased from Sigma-Aldrich and dissolved in water and sterilized in the same way to produce stock concentrations of 500 mg and 5 mM, respectively

To produce GMMA, the strains were grown in medium supplemented with antibiotics (30 μg/mL kanamycin for Ss; 30 μg/mL kanamycin and 100 μg/mL erythromycin for Sf2a) to OD_600nm = 0.3. The cultures were then diluted into 1 L of fresh medium (without antibiotics) to a starting OD_600nm of 0.05 and incubated at 37 °C. After growing to OD_600nm of 0.4, in case of Ss, and of 0.3, in case of Sf2a, cultures were centrifuged for 15 min at 5000 × g and the supernatants removed for GMMA preparation.
The bacterial pellets were solubilized after two washes with cold phosphate buffered saline (PBS) by resuspending in lysis buffer (0.8 M urea, 0.4% SDS, 1 mM DTT and 20 mM Tris, pH 7.4) to prepare lysates.
The culture supernatants were filtered through a 0.22 μm filter unit (Millipore). The filtered samples were concentrated in a Stirred Cell Model 8400 (Millipore) through a 100,000 Da regenerated cellulose membrane (Millipore). GMMA were then separated from soluble proteins by ultracentrifugation at 186,000 × g for 2 h at 4 °C (Optima™ L-series, 45Ti rotor, Beckman Instruments). The GMMA pellets were washed once with cold PBS, resuspended in 20 mM Tris, pH 7.4, then filtered through a 0.22 μm filter.