Saccharomyces cerevisiae mannan

Functional activity of the CP, LP and AP was determined essentially as described (16 (link)). In short, Nunc-Maxisorb ELISA plates were coated overnight to specifically activate the CP (coated with 3 μg/ml human IgM (Calbiochem)), LP (coated with 20 μg/ml Saccharomyces cerevisiae mannan (Sigma)), or AP (coated with 20 μg/ml Salmonella enteriditis LPS (Sigma)). Plates were blocked with 1% (w/v) BSA in PBS with 0.05% (v/v) Tween 20 (Merck). The indicated percentages of NHS or mouse serum (Innovative Research) were incubated with 1 μM of recombinant S. aureus proteins in the appropriate assay buffers for a maximum of 5 min at 25 °C (veronal-buffered saline (VBS) at pH 7.5 with 0.1% (w/v) gelatin, 500 μM CaCl₂, and 250 μM MgCl₂ for CP and LP; VBS at pH 7.5 with 0.1% (w/v) gelatin, 5 mM MgCl₂, and 10 mM EGTA for AP). Deposited C3b, C4b, and C5b-9 were detected with specific antibodies (0.1 μg/ml α-human C3c WM-1 clone digoxigenin (DIG) labeled or 1 μg/ml rat-α-mouse C3 (Hycult, HM1078), American Type Culture Collection; 1 μg/ml αC4d, Quidel; 1 μg/ml αC5b-9 aE11, Santa Cruz respectively). Secondary, HRP-labeled antibodies were detected with 100 μg/ml tetramethylbenzidine and 60 μg/ml ureum peroxide in 100 mM sodium acetate buffer at pH 6.0. The reaction was stopped by adding an equal volume of 2 M H₂SO₄, and the absorbance at 450 nm was measured using a BioRad microplate reader.

Bt was cultured anaerobically at 37 °C in minimal media containing an appropriate carbon source. or in TYG (tryptone yeast extract glucose medium) as described previously21 (link). Growth curves presented in the paper are averages of six biological replicates. Bt was grown in 5 mL minimal media on 0.5% w/v Saccharomyces cerevisiae mannan (Sigma) or glucose as the sole source to mid exponential phase (OD_600nm 0.6-0.8). Cells were harvested by centrifugation, 5000 x g 10min at room temperature and washed in 5 ml Phosphate buffered Saline pH 7.1 (PBS) before being resuspended in 500 µL PBS. Cells (50 µL) were assayed against yeast mannan (10 mg ml^-1) at 37 °C for 16 h. Assays were analysed by thin layer chromatography, 5 µL of each sample was spotted onto silica plates and resolved in butanol/acetic acid/water buffer. The plates were dried and sugars visualised by orcinol/sulphuric acid heated to 70°C.