The structure of Bordetefla pertussis 1414 lipid A was investigated by classical methods of chemical analysis as well as plasma desorption mass spectrometry and fast atom bombardment mass spectrometry. Previous analysis showed that it contained a bisphosphorylated II-(1-6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkage. The presence of two main molecular species as seen by thin-layer chromatography was confirmed by plasma desorption mass spectrometry, in which the larger signal was attributable to a molecular ion containing two glucosamine, two phosphate, one tetradecanoic acid, one hydroxydecanoic acid, and three hydroxytetradecanoic acid residues. The ion of the smaller signal was lighter by the mass of one hydroxytetradecanoic acid residue (226 Da). The fatty acids in ester linkage were localized by chemical and fast atom bombardment mass spectrometry analysis. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site for Kdo (3-deoxy-Dmanno-octulosonic acid).Endotoxins are major constituents of the outer membrane of gram-negative bacteria. Their main interest lies in the many biological activities they induce even at low doses. They are ensembles of related lipopolysaccharides (LPS) consisting of a lipid moiety, termed lipid A, linked to a polysaccharide chain of variable length.The biological activities and serological relationships of the endotoxins of various Bordetella species have been explored in the light of the variations in the diseases they cause (1, 35).Recently, the relationship between Bordetella parapertussis and Bordetella bronchiseptica was clarified by the finding that their O chains were identical although their core regions were different (13). The serological properties of the rough-type endotoxin of Bordetella pertussis, the agent of whooping cough, have been well documented, and the determination of its complex glycosidic structure is nearing completion (2,5,12,14,20,22,27).The lipid moiety of B. pertussis LPS, like that of other endotoxins, is responsible for most of the biological activities of the molecule (11). Early work indicated a backbone structure identical to that of enteric bacteria, i.e., a B-1i',6-linked diglucosamine phosphorylated on the a-glycosidic and 4' carbons and the two glucosamines amidated by 3-hydroxytetradecanoic acid (9, 15). However, it differs from the lipid A of enteric organisms in that it contains only two or three esterlinked fatty acids, instead of three to five, and one of those is hydroxydecanoic acid (8,11). With this information, it was possible, by the use of plasma desorption mass spectrometry (PDMS), to determine how many molecular species were present in a preparation and the composition and relative abundance of each (18). These results are presented here together with nuclear magnetic resonance (NMR) confirmation of the C-4' position of the glucosamine II The B. pertussis endotoxin used in this work was prepared by hot phenol-water extraction...

Structural and immunological differences between the two components that are usually present in unequal quantities in Bordetella pertussis endotoxin preparations and are visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been studied by using strains 1414, A100, and 134, all in phase I. According to analyses by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer chromatography, the minor (8%) component of the endotoxin of strain 1414 (endotoxin 1414) appeared to be the predominating component of endotoxins A100 and 134. The masses of the carbohydrate chains isolated from endotoxin A100 and from the major component of endotoxin 1414 were 1,649 and 2,311 atomic mass units, respectively, as determined by 252Cf plasma desorption mass spectrometry. Comparison of the 1H nuclear magnetic resonance spectra of these chains established that four N-acetyl groups, an N-methyl group, and a 6-deoxy function, which characterize the nonreducing, distal trisaccharide of the glycose chain of strain 1414, were absent from that of strain A100. The antigenicity of endotoxin 1414, as measured by enzyme-linked immunosorbent assay, was higher than that of endotoxin A100, but fell below it when the glycose chain of endotoxin 1414 was deprived of seven sugars by treatment with nitrous acid. This observation suggests that at least three (distal, proximal, and intermediate) regions of the glycose chain of endotoxin 1414 carry antigenic determinants. One of these, located in the distal trisaccharide, is absent from both endotoxins A100 and 134.