Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution

Abstract

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...

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis

“…This observation is consistent with the high LPS heterogeneity that was previously reported for other Bordetella strains (10,65) and other gram-negative bacteria (reviewed in reference 11). Since the enzymes that cooperate in the lipid A biosynthesis route are thought to be interdependent and to work in a preferred order (reviewed in reference 59), it is hard to imagine how significant lipid A heterogeneity could arise during biosynthesis.…”

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

“…The spectrum revealed structural differences from that of the B. pertussis lipid A. The heterogeneous spectrum, however, did give mass peaks corresponding to molecular species found in other Bordetella species, such as B. bronchiseptica (29) and B. hinzii (30). The detailed structure will be described in a future publication.…”

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

“…In mild conditions, the acyloxyacyl ester could be released without splitting the secondary ester linkage between the two fatty acids and characterized by GC-MS. These conditions have been used to determine the structures of many lipid A preparations, such as those of Bordetella (12)(13)(14)(15), Helicobacter (16), and Yersinia (17). We also demonstrated the importance of such a sequential release in the case of Yersinia lipid A structure previously erroneously described as being identical to that of Escherichia coli.…”

“…Cells were grown as described (13,(19)(20)(21)(22) Briefly, Citrobacter cells were grown to late exponential phase in a brain-heart infusion (Difco) at 37jC under constant aeration in a New Brunswick 25 liter fermenter.…”

“…These structures were notable for their peculiarly high variability among species and even strains. In B. bronchiseptica, lipid A structural variability has been attributed to relaxed enzyme specificity (6,13,15). The lipids A of two human pathogens, B. pertussis and B.…”