The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated 1(1'-+6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid Meningococcal infections represent a serious problem for small children and young adults in many parts of the world, the lethality being high even in developed countries (1, 5, 6). The causative agent, Neisseria meningitidis, like other gramnegative bacteria, possesses an endotoxin complex located in the bacterial outer cell membrane. Endotoxin (lipopolysaccharide [LPS]) is known to cause or to contribute to the severe symptoms of meningitis, culminating in irreversible shock (37). Meningococci vary in the ability to release endotoxin-containing outer membrane vesicles into the surroundings, both in vitro and in vivo (7). Recent studies performed with N. meningitidis isolates from the nasopharynx of carriers and from cerebrospinal fluid and blood samples from patients with meningococcal infection demonstrated that overproduction and liberation of endotoxin are strongly associated with pathogenicity, regardless of the serological group or serotype of the strain (8, 9).Lipid A represents the principal structural component responsible for the harmful biological activities of LPS (32). To understand the structural parameters required for the expression of endotoxic activity, it appears important to know the precise chemical structure of the lipid A component of pathogenic gram-negative bacteria such as N. meningitidis. The present paper elucidates the primary structure of the lipid A component of N. meningitidis isolated from a strain (M986-NCV1) expressing the serogroup B LPS. It will be shown that this lipid A shares certain structural features with the well-studied enterobacterial lipid A but that it differs significantly in the substitution of the hydrophobic backbone and in the nature and locations of acyl chains. * Corresponding author. MATERIALS AND METHODSMicroorganism and LPS. N. meningitidis M986-NCV1, a nonencapsulated variant of the group B strain M986, was obtained from the culture collection of the Center for Biologics Evaluation and Research. This strain was grown overnight in tryptic soy broth (Difco Laboratories, Detroit, Mich.) in 2.8 liters of baffled Fernbach ...
The structure of the lipid A component of lipopolysaccharides isolated from two wild-type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas-liquid chromatography/mass spectrometry, laser-desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic /31,6-linked Dglucosamine disaccharide [P-~-GlcpN-(l+ 6)-~-GlcpN], phosphorylated in positions 4 and 1. Position 6' of the P-~-GlcpN-(l + 6)-~-GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C-4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide-bound 3-0-acylated (R)-3-hydroxydodecanoic acid groups [12 : O(3-OH)I at positions 2 and 2' of the GlcN dissacharide and two ester-bound (R)-3-hydroxydecanoic acid groups [lo: O(3-OH)] at positions 3 and 3'. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3-OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa-and pentaacyl lipid A the 3-hydroxyl group of the two amide-linked 12 : O(3-OH) residues are acylated by either dodecanoic (12: 0) or (S)-2-hydroxydodecanoic acid [12: 0(2-OH)], the lipid A species with two 12:0(2-OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide.Pseudomonas aeruginosa is an ubiquitous Gram-negative bacterium which colonizes and infects preferentially hospitalized and immunocompromized patients [ 11. In particular, P. aeruginosa is the most frequent pathogen isolated from patients with cystic fibrosis and burns [2-41. In the pathogenic potential of P. aeruginosu the cell wall lipopolysaccharide (endotoxin) is of great importance [5]. This is suggested, in particular, by the toxic properties of lipopolysaccharide Ahhreviutions. LPS, lipopolysaccharide; dOclA, 3-deoxy-Dmanno-octulosonic acid; CI-MS, ET-MS and LD-MS, chemicdl-ionization, electron-impact and laser-desorption mass spectrometry; GlcN, 2-amino-2-deoxy-u-glucose; GlcpN, 2-amino-2-deoxy-~-glucopyranosc; GlcNAc, 2-acetamido-2-deoxy-~-glucose; GlcNAc-ol, 2-acetamido-2-deoxy-~-glucitol; GalN, 2-amino-2-deoxy-~-galactose; P-~-GlcpNAc-(l + 6)-~-GlcpNAc, 2-acetamido-6-0-(2-acetamido - In order to gain knowledge on the chemical determinants involved in endotoxic activity, the chemical architecture of the lipid A component of P. aeruginosa lipopolysaccharides which are less toxic than enterobacterial preparations [5] has been studied by various groups and some of its structural features were elucidated 15, 10, 11,[16][17][18][19][20][21][22][23][24][25][26]. Also, structural proposals of P. aeruginosa lipid A have been mad...
Non-instrumental immunoassay methods based on immunofiltration and microtiter particle agglutination (MPA) techniques have been developed using coloured polyacrolein latex. These methods have been applied to the quantification of the group-specific polysaccharide, A-PS, of S.pyogenes (group A Streptococcus) and compared to the standard ELISA tests. The assay with the ability to detect the lowest concentration of antigen was MPA; as little as 0.05 ng A-PS/ml or 10(4) cells/ml could be detected in 1.5 h. In comparison to ELISA test the sensitivity of MPA was 10 times higher and the procedure of the assay was much more simple. The sensitivity of the immunofiltration assay using both enzyme and latex markers was shown to be the same (50 ng A-PS/ml) and the duration of the assay 3-5 min. No cross-reactions of latex conjugates with non A Streptococcus cell lysates have been observed. The developed methods are easy to perform and require neither sophisticated equipment nor specially trained personal.
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