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.
Bacterial infections are a major cause of morbidity and mortality worldwide and the rise of antibiotic resistance necessitates development of alternative treatments. Pathogen adhesins that bind to host cells initiate disease pathogenesis and represent potential therapeutic targets. We have shown previously that the BspC adhesin in Group B Streptococcus (GBS), the leading cause of bacterial neonatal meningitis, interacts with host vimentin to promote attachment to brain endothelium and disease development. Here we determined that the BspC variable (V-) domain contains the vimentin binding site and promotes GBS adherence to brain endothelium. Site directed mutagenesis identified a binding pocket necessary for GBS host cell interaction and development of meningitis. Using a virtual structure-based drug screen we identified compounds that targeted the V-domain binding pocket, which blocked GBS adherence and entry into the brain in vivo. These data indicate the utility of targeting the pathogen-host interface to develop anti-virulence therapeutics.
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.
Background RhD‐negative blood products are in chronic short supply leading to renewed interest in utilizing RhD‐positive blood products for emergency transfusions. This study assessed parental perceptions of emergency RhD‐positive blood use in children. Methods A survey of parents/guardians was conducted on their tolerance of transfusing RhD‐positive blood to RhD‐negative female children ≤17 years old at four level 1 pediatric hospitals. Results In total, 621 parents/guardians were approached of whom 378/621 (61%) completed the survey in its entirety and were included in the analysis. Respondents were mostly females [295/378 (78%)], White [242/378 (64%)], had some college education [217/378 (57%)] and less than $60,000 annual income [193/378 (51%)]. Respondents had a total of 547 female children. Most children's ABO [320/547 (59%)] and RhD type [348/547 (64%)] were not known by their parents; of children with known RhD type, 58/186 (31%) were RhD‐negative. When the risk of harm to a future fetus was given as 0–6%, more than 80% of respondents indicated that they were likely to accept RhD‐positive blood transfusions on behalf of RhD‐negative female children in a life‐threatening situation. The rate of willingness to accept emergent RhD‐incompatible blood transfusions significantly increased as the potential survival benefit of the transfusion increased. Conclusion Most parents were willing to accept RhD‐positive blood products on behalf of RhD‐negative female children in an emergency situation. Further discussions and evidence‐based guidelines on transfusing RhD‐positive blood products to RhD‐unknown females in emergency settings are needed.
Streptococcus agalactiae (Group B Streptococcus , GBS), is an opportunistic pathogen capable of causing invasive disease in susceptible individuals including the newborn. Currently GBS is the leading cause of meningitis in the neonatal period. We have recently shown that GBS interacts directly with host type III intermediate filament vimentin to gain access to the central nervous system. This results in characteristic meningeal inflammation and disease progression; however, the specific role of vimentin in the inflammatory process is unknown. Here we investigate the contribution of vimentin to the pathogenesis of GBS meningitis. We show that a CRISPR targeted deletion of vimentin in human cerebral microvascular endothelial cells (hCMEC) reduced GBS induction of neutrophil attractants IL-8 and CXCL-1, as well as NFκB activation. We further show that inhibition of vimentin localization also prevented similar chemokine activation by GBS. One known chemokine regulator is the nucleotide-binding oligomerization domain containing protein 2 (NOD2), which is known to interact directly with vimentin. Thus, we hypothesized that NOD2 would also promote GBS chemokine induction. We show that GBS infection induced NOD2 transcription in hCMEC comparable to the muramyl dipeptide (MDP) NOD2 agonist, and the chemokine induction was reduced in the presence of a NOD2 inhibitor. Using a mouse model of GBS meningitis we also observed increased NOD2 transcript and NOD2 activation in brain tissue of infected mice. Lastly, we show that NOD2 mediated IL8 and CXCL1 induction required vimentin, further indicating the importance of vimentin in mediating inflammatory responses in brain endothelium.
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