Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. less than or equal to 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 [C-14]acetate yielded C-14-lipooligosaccharides containing similar to 600 cpm/ng. Gel sieving resolved C-14-lipooligosaccharide-containing aggregates with an estimated molecular mass of greater than or equal to 20 x 10(6) Da (peak A) and similar to1 x 106 Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS polyacrylamide gel electrophoresis profile. C-14-Labeled capsule copurified with 14C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only C-14-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.