title: Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B.
abstract: Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBP-dependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin.sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.
authors.affiliation: The Buck Institute for Age Research, Novato, California 94945, USA.
authors.forename: Deborah M B
authors.initials: DM
authors.lastname: Post
authors.forename: DeSheng
authors.initials: D
authors.lastname: Zhang
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Eastvold
authors.forename: Athmane
authors.initials: A
authors.lastname: Teghanemt
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
authors.forename: Jerrold P
authors.initials: JP
authors.lastname: Weiss
mesh_terms: D000209:Acute-Phase Proteins / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000954:Antigens, Surface / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry; D006801:Humans; D007249:Inflammation; D008055:Lipids / Q000737:chemistry; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000737:chemistry; D049449:Luminescence; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D009345:Neisseria meningitidis / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D004731:Endotoxins; D005227:Fatty Acids; D008055:Lipids; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D051197:Toll-Like Receptor 4; D000099231:Lipopolysaccharide-Binding Protein