title: Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions.
abstract: To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.
authors.affiliation: Department of Microbiology, The Inflammation Program, University of Iowa and Veterans' Administration Medical Center, Iowa City, Iowa 52242, USA.
authors.forename: P C
authors.initials: PC
authors.lastname: Giardina
authors.forename: T
authors.initials: T
authors.lastname: Gioannini
authors.forename: B A
authors.initials: BA
authors.lastname: Buscher
authors.forename: A
authors.initials: A
authors.lastname: Zaleski
authors.forename: D S
authors.initials: DS
authors.lastname: Zheng
authors.forename: L
authors.initials: L
authors.lastname: Stoll
authors.forename: A
authors.initials: A
authors.lastname: Teghanemt
authors.forename: M A
authors.initials: MA
authors.lastname: Apicella
authors.forename: J
authors.initials: J
authors.lastname: Weiss
mesh_terms: D000085:Acetates / Q000378:metabolism*; D000209:Acute-Phase Proteins; D016667:Bacterial Capsules; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism*; D005227:Fatty Acids / Q000032:analysis; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008954:Models, Biological; D009345:Neisseria meningitidis / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000085:Acetates; D000209:Acute-Phase Proteins; D001427:Bacterial Toxins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D004731:Endotoxins; D005227:Fatty Acids; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000099231:Lipopolysaccharide-Binding Protein; C023023:lipid-linked oligosaccharides