Identification of a novel cationic glycolipid in Streptococcus agalactiae that contributes to brain entry and meningitis

Abstract

Bacterial membrane lipids are critical for membrane bilayer formation, cell division, protein localization, stress responses, and pathogenesis. Despite their critical roles, membrane lipids have not been fully elucidated for many pathogens. Here, we report the discovery of a novel cationic glycolipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), which is synthesized in high abundance by the bacterium Streptococcus agalactiae (Group B Streptococcus, GBS). To our knowledge, Lys-Glc-DAG is more positively charged than any other known lipids. Lys-Glc-DAG carries 2 positive net charges per molecule, distinct from the widely described lysylated phospholipid lysyl-phosphatidylglycerol (Lys-PG) that carries one positive net charge due to the presence of a negatively charged phosphate moiety. We use normal phase liquid chromatography (NPLC) coupled with electrospray ionization (ESI) high-resolution tandem mass spectrometry (HRMS/MS) and genetic approaches to determine that Lys-Glc-DAG is synthesized by the enzyme MprF in GBS, which covalently modifies the neutral glycolipid Glc-DAG with the cationic amino acid lysine. GBS is a leading cause of neonatal meningitis, which requires traversal of the endothelial blood–brain barrier (BBB). We demonstrate that GBS strains lacking mprF exhibit a significant decrease in the ability to invade BBB endothelial cells. Further, mice challenged with a GBSoΔmprF mutant developed bacteremia comparably to wild-type (WT) infected mice yet had less recovered bacteria from brain tissue and a lower incidence of meningitis. Thus, our data suggest that Lys-Glc-DAG may contribute to bacterial uptake into host cells and disease progression. Importantly, our discovery provides a platform for further study of cationic lipids at the host–pathogen interface.

Insights into the role of lipoteichoic acids and MprF function in Bacillus subtilis

“…The LTA profiles observed in B. subtilis and S. aureus lacking MprF (seen as shorter and/or decreased in abundance) and the overexpression of B. subtilis MprF (causing a smeared & retarded mobility of the LTA) ( Fig 4B, 6A ) suggest that MprF positively regulates LTA production ( Fig 8A ). Other studies have indicated that MprFs may have relaxed substrate specificities across species (22, 62) and even synthetise lysyl-glucosyl-DAG glycolipid in Streptococcus agalactiae (63). Recently, it has been shown that when S. aureus LtaS uses PGol as an alternative anchor to the glycolipid produced by UgtP, the concentration of these free lipid starter units regulates the length of the LTA polymerised in vitro (33).…”

Gram-positive bacterial membrane lipids at the host–pathogen interface

“…Aminoacylation is not specific to PG and in the case of Listeria , Lys-CL is observed [ 10 ]. Furthermore, the amino-acylated glycolipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG) was recently identified in Streptococcus agalactiae ( Table 1 ) [ 11 ]. In general, amino-acylation aids in masking the negative charge associated with the extracellular surface of the membrane that is attributed to the phosphate headgroup of phospholipids, which has beneficial implications for human pathogens.…”

“…S . aureus Δ mprF strains are significantly attenuated in blood survival in vivo, whereas Enterococcal and Streptococcal strains were not [ 8 , 11 , 13 ]. Removal of Lys-PG from the membrane of Staphylococci and Enterococci resulted in a significant reduction of the negative extracellular surface charge creating a more neutral surface charge, which decreased susceptibility to CAMPs, antibiotics, and opsonophagocytic killing by neutrophils [ 8 , 13 , 14 ].…”

“…Removal of both Lys-PG and Lys-Glc-DAG from the membrane of S . agalactiae led to reduced growth in low pH conditions and significantly less invasion of human brain endothelial cells in vitro but no change in susceptibility to CAMPs and other antimicrobials [ 11 , 15 ]. S .…”

“…S . agalactiae Δ mprF strains exhibited similar bloodstream survival as WT strains; however, the pathogenesis of meningitis and blood–brain barrier (BBB) crossing was significantly attenuated suggesting the lysine lipids are important for interaction with the BBB [ 11 ]. While Staphylococci may use Lys-PG to resist neutrophil killing during bloodstream infections, Enterococci and Streptococci may utilize LTA as the major factor to survive neutrophil defenses [ 15 ].…”