Vehicle controls and chemical inhibitors.

For each experiment involving chemical inhibitors, sterile dimethyl sulfoxide (DMSO; Sigma) was used as the chemical solvent and experimental vehicle control. SB203580 (Cell Signaling) was used to inhibit p38 MAPK activity, and SP600125 (Cell Signaling) was used to inhibit JNK activity. Z-VAD-fmk (ApexBio) was used as a general caspase inhibitor, and staurosporine (Cell Signaling), which is a potent protein kinase inhibitor that induces apoptosis, was used as a positive control for caspase 3/7 activation and induction of apoptosis. Necrostatin-1 (Sigma-Aldrich) was used to inhibit RIPK1 kinase, and GSK872 (Calbiochem) was used to inhibit RIPK3. Each inhibitor (except staurosporine) was incubated with each of the GBS strains used in this study at time points and drug concentrations matching experimental conditions to determine effects on GBS growth and viability (data not shown). Following incubation in liquid media, bacterial samples were collected from each sample/condition, plated on Todd-Hewitt agar, and grown overnight at 37°C in 5% CO₂ to quantify CFU/ml from the liquid cultures as conducted previously (49).