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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.

The endotoxin (lipopolysaccharide) of Bordetella pertussis, the agent of whooping cough, consists of a lipid A linked to a highly branched dodecasaccharide containing several acid and amino sugars. The elucidation of the polysaccharide structure was accomplished by first analyzing the structures of fragments obtained by hydrolysis and nitrous deamination and then piecing the fragments together. The fine structure of the antigenic distal pentasaccharide, presented here, was determined by chemical analyses as well as by high-resolution nuclear magnetic resonance and mass spectrometry. The complete structure was reconstituted and confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The following structure was derived from the combined experimental data:The detailed structure combined with previously reported serological data now allows the synthesis of its epitopes for potential vaccines. ß

The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C-2P P: this was dodecanoic acid (C 12 ) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C 14 ) in two other Y. enterocolitica and hexadecenoic acid (C 16:1 ) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.z 2000 Federation of European Biochemical Societies.

The structures of lipids A isolated from the lipopolysaccharides (LPSs; endotoxins) of three different pathogenic Bordetella bronchiseptica strains were investigated by chemical composition and methylation analysis, gas chromatography-mass spectrometry, nuclear magnetic resonance, and plasma desorption mass spectrometry (PDMS). The analyses revealed that the LPSs contain the classical lipid A bisphosphorylated ␤-(136)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Their structures differ from that of the lipid A of Bordetella pertussis endotoxin by the replacement of hydroxydecanoic acid on the C-3 position with hydroxydodecanoic acid or dodecanoic acid and the presence of variable amounts of hexadecanoic acid. The dodecanoic acid is the first nonhydroxylated fatty acid to be found directly linked to a lipid A glucosamine. The lipids A were heterogeneous and composed of one to three major and several minor molecular species. The fatty acids in ester linkage were localized by PDMS of chemically modified lipids A. B. pertussis lipids A are usually hypoacylated with respect to those of enterobacterial lipids A. However, one of the three B. bronchiseptica strains had a major hexaacylated molecular species. C-4 and C-6 hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site of the polysaccharide. The structural variability seen in these three lipids A was unusual for a single species and may have consequences for the pathogenicity of this Bordetella species.Bordetella bronchiseptica is a respiratory tract pathogen of mammals causing a whooping cough syndrome milder than that due to Bordetella pertussis in humans. This bacterium causes bronchopneumonia in rabbits and guinea pigs, kennel cough in dogs, and atrophic rhinitis in piglets (13). Recently, it has been described as responsible for bronchopneumonia in human immunosuppressed patients (1, 27, 28) and in at least two immunocompetent patients, one of whom was exposed to infected animals (14). Its endotoxin, a mixture of lipopolysaccharides (LPSs), which are a major component of the bacterial outer membrane, has been implicated as a virulence factor. B. bronchiseptica, like Bordetella parapertussis, produces smoothtype LPSs. Their O chains have identical structures (11). B. pertussis LPSs, which are of the rough type, have been analyzed, and their structures have recently become available (4,6,25). Structural variability in parts of LPS other than the O chains may thus relate to pathogenicity or to host specificity. We report here the detailed structural characterization of the lipid A moieties of three different strains of B. bronchiseptica grown under the same culture conditions and show that they have greater variability than that usually encountered in a single species. MATERIALS AND METHODSMass spectrometry. Plasma desorption mass (PDM) spectra were obtained with a Depil TOF 21 mass spectrometer (10). Flight distance was 90 cm, acceleration voltage was ϩ15 or Ϫ15 kV, an...

Representative strains of Bordetella bronchiseptica and B. parapertussis were found to produce smooth lipopolysaccharides (LPS) having identical antigenic O-polysaccharide components composed of linear unbranched polymers of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-L-galacto-pyranosyluronic acid residues. These LPSs differed from the LPS of B. pertussis which produces only rough-type LPS, devoid of O-polysaccharide. While B. bronchiseptica and B. parapertussis had chemically and immunologically identical O-polysaccharide structures, their core oligosaccharide components differed. The core oligosaccharide of B. parapertussis was chemically distinct from the core of B. bronchiseptica which appeared to be structurally and immunologically similar to a core oligosaccharide of B. pertussis.