Membrane Permeability Assay

Changes in the bacterial cell membrane permeability was assessed by propidium iodide (PI) (Sigma) uptake and the metabolic activity by calcein AM (BioLegend) staining essentially as described (Cho and Lee, 2011; Ohsumi et al., 2015). PI enters only membrane-compromised cells and fluoresces in the red spectrum when binding to nucleic acids within the cells (Veerman et al., 2004). On the other hand, calcein AM diffuses passively into the cytoplasm, where it is converted into green fluorescent calcein via native esterases. Calcein fluorescence is retained in live cells but leaks out when the plasma membrane is compromised. An overnight culture of S. mutans was resuspended in BHI to an OD_600nm = 0.3. The bacteria were then treated with different concentrations of CBG (0, 1.25, 2.5, 5, 8, and 10 μg/ml) for 2 h at 37°C. At the end of incubation, the bacteria were stained with 10 μg/ml PI and 10 μg/ml calcein AM for 20 min at 37°C, followed by flow cytometry (BD LSR-Fortessa flow cytometer, BD Biosciences) using the 488 nm excitation laser and collecting the data using the red and green filters, respectively.