Group B streptococcal membrane vesicles induce proinflammatory responses in neonatal meninges

Streptococcus agalactiae (Group B Streptococcus; GBS) is the leading cause of neonatal meningitis, resulting in morbidity and lasting neurological effects in survivors. GBS makes three unique glycolipids in their cell membrane, which we have previously shown to promote bloodstream survival and meningitis. GBS also produces membrane vesicles (MVs), but the lipid content is not known, nor is it known how glycolipids impact MV biogenesis, cargo, and function. Additionally, it is currently unknown how GBS MVs may contribute to systemic infection, meningitis, and resulting neuroinflammation. Here, using our isogenic lipid mutants in a clinically relevant sequence type 17 and capsule type III strain, we identify that the MV lipidome replicates that of the parent bacterial cell, and that MVs isolated from a glycolipid-devoid strain have an altered MV proteome. Investigating the impact of GBS MVs on brain endothelial cells in vitro, we observe significant increases in proinflammatory chemokines CXCL-1 and IL-6 from MVs isolated from WT and lipid mutant strains. Furthermore, we show that the proteins on the MV surface contribute to cytokine induction, which is dependent on Toll-like receptor 2 (TLR2)-mediated signaling. Finally, using a neonatal murine infection model, we observe significant increases in proinflammatory chemokines CXCL-1, IL-6, TNFα, and IL-1β in the murine leptomeninges and brain when GBS MVs are present in the cerebrospinal fluid. This work reveals unexplored functions of bacterial glycolipids in MV biogenesis and identifies GBS MVs as potent inflammatory molecules to the meninges and brain endothelium that may contribute significantly to meningitis disease progression.