The structure of the core-lipid A region of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica has been analyzed. Lipopolysaccharides were deacylated using strong alkaline hydrolysis, the products were separated by high performance anion-exchange chromatography and analyzed by NMR and mass spectrometry. The following structure of the products can be deduced from the experimental results: Bacteria of the genus Bordetella are gram-negative aerobic coccobacilli, assigned to the family Alcaligenaceae. They are found only in warm-blooded animals. Bordetella pertussis and Bordetella parapertussis are human respiratory tract pathogens, Bordetella bronchiseptica and Bordetella hinzii are animal pathogens but are increasingly found in AIDS and cystic fibrosis patients [1±4]. Whereas B. pertussis produces R-type lipopolysaccharide (LPS) without the polysaccharide O-chain, B. bronchiseptica and B. hinzii LPSs have O-chains. B. parapertussis and B. bronchiseptica produce an O-chain with the same repeating unit [5]. The structure of the core part of Bordetella LPS has been partially determined only for B. pertussis [6]. Here we present the structure of the carbohydrate backbone of the LPS from B. bronchiseptica and B. hinzii.
E X P E R I M E N T A L P R O C E D U R E SBacterial strains and lipopolysaccharide isolation B. bronchiseptica strain 110H was obtained from D. Bemis (University of Tennessee, Knoxville, TN, USA) and was originally isolated from a dog in New York in 1970. B. bronchiseptica strain Bp512 (formerly NCTC 8762) was obtained from James Musser (NIH, NIAID, Rocky Mountain Labs, Hamilton, MT, USA) and was originally isolated from the upper respiratory tract of a human in Manchester, UK in 1950. B. hinzii strain ATCC 51730 was originally isolated from the blood of an AIDS patient. Cells were cultivated and LPS isolated as described previously [5].NMR spectroscopy and general methods 1 H and 13 C NMR spectra were recorded using a Varian Inova 500 spectrometer in D 2 O solutions at 25 8C with acetone standard (2.225 p.p.m. for 1 H and 31.5 p.p.m. for 13 C) using vnmr 6.1b software pulse sequences gDQCOSY, TOCSY (mixing time 120 ms), NOESY (mixing time 200 ms), gHSQC, gHMBC (optimized for 5 Hz coupling constant), gHSQC-TOCSY, digital resolution in F2 was 1.2 Hz´point 21X31 P and 1 H± 31 P HMQC (optimized for 7 Hz coupling constant) spectra were recorded on a Varian Inova 400 spectrometer. Spectra were assigned with the help of the pronto program [7]. Electrospray mass spectra were obtained using a Micromass Quattro spectrometer in 50% MeCN with 0.2% HCOOH at a flow rate of 15 mkl´min 21 with direct injection. GLC, GLC-MS, methylation and monosaccharide analyzes were performed as described previously [8].
Preparation of oligosaccharidesLPS from each bacterial strain (200 mg) was heated at 100 8C for 4 h in 4 m NaOH (4 mL) containing a small amount of NaBH 4 , cooled and neutralized to pH 8±9 with 2 m HCl (7.5 mL). The precipitate was removed by centrifugation and the products were separated...