Identification of a novel cationic glycolipid inStreptococcus agalactiaethat contributes to brain entry and meningitis

Abstract

Streptococcus agalactiae (Group B Streptococcus; GBS) is the etiological agent of meningitis and severe invasive diseases in newborns. The cellular membrane, a critical site for host-pathogen interactions, is poorly characterized in GBS. Here, we analyzed the GBS lipidome using liquid chromatography coupled with electrospray ionization tandem mass spectrometry. We identified a novel amino-acylated glycolipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), which is abundant in all GBS strains analyzed. Through heterologous expression and gene deletion, we demonstrate that the GBS multiple peptide resistance factor (MprF) synthesizes Lys-Glc-DAG, as well as lysyl-phosphatidylglycerol (Lys-PG), which has been characterized in many pathogenic bacteria. Consistent with a critical role in host-GBS interactions, the GBSΔmprF mutant exhibited a reduction of surface net negative charge and a significantly reduced ability to invade human cerebral microvascular endothelial cells. Further, mice challenged with the GBSΔmprF mutant developed bacteremia comparably to wild-type GBS-challenged mice, yet exhibited fewer bacterial counts in the brain and less meningeal inflammation. Overall, we demonstrate that GBS has uniquely evolved a multifunctional MprF enzyme that results in the synthesis of a major cationic membrane glycolipid and plays a significant role in meningitis pathogenesis. Moreover, our results illustrate the novel insights obtained from comprehensive lipidomic profiling of major human pathogens.

Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum

“…However, these methods lack molecular specificity and sensitivity for the comprehensive characterization of membrane lipids [20]. The phospholipids phosphatidylglycerol (PG) and cardiolipin (CL), and the glycolipids monohexosyldiacylglycerol (MHDAG) and dihexosyldiacylglycerol (DHDAG) were previously detected in all three streptococcal species [14][15][16][17][18][19][21][22][23].…”

Streptococcus pneumoniae,S. pyogenes, andS. agalactiaemembrane phospholipid remodeling in response to human serum

“…Interestingly, PC is observed in the membrane of GAS when defined medium is supplemented with 5% v/v human serum, indicating that human serum, but not Todd-Hewitt Broth or defined medium, provide substrates required for PC biosynthesis (Supplemental Figure S2). The ESI/MS of the major PC species identified in the GAS membrane is shown in Supplemental Figure S3 Figure S2) (23). The lipidomic profile of the GBS is unchanged when cultured in defined medium ( Table 1).…”

“…The lipid profile of GBS cultured in Todd-Hewitt Broth consists of MHDAG, DHDAG, PG, and CL, as well as the aminoacylated PG molecule lysyl-PG (Lys-PG), and a recently described novel, cationic glycolipid, Lysyl-Glucosyl-DAG (LGD) (Table 1, Supplemental Figure S2) (23). The lipidomic profile of the GBS is unchanged when cultured in defined medium (Table 1).…”

“…However, these methods lack molecular specificity and sensitivity for the comprehensive characterization of membrane lipids (20). The phospholipids phosphatidylglycerol (PG) and cardiolipin (CL), and the glycolipids monohexosyldiacylglycerol (MHDAG) and dihexosyldiacylglycerol (DHDAG) were previously detected in all three streptococcal species (14)(15)(16)(17)(18)(19)(21)(22)(23).…”

“…It is currently unknown whether GBS scavenge lyso-pPC or pPC from human serum or if it is de novo synthesized from PC. This figure combines lipids and genes described in literature(14)(15)(16)(17)(18)(19)(21)(22)(23)(24)(25)32,33) and detected lipids from Table1.…”