Occurrence of 2-keto-3-deoxy-D-manno-octonic acid in lipopolysaccharides isolated from Vibrio parahaemolyticus

Abstract

The occurrence of 2-keto-3-deoxy-D-manno-octonic acid (KDO) in lipopolysaccharides (LPS) of Vibrio parahaemolyticus was demonstrated for the first time by gas chromatography-mass spectrometry after dephosphorylation, reduction, and methylation. KDO was virtually completely phosphorylated, since no KDO was detected by either gas chromatography or thiobarbituric acid assay before dephosphorylation. The level of KDO in all six strains of V. parahaemolyticus investigated ranged from 0.37 to 0.69%, which was considerably lower than that in enterobacterial LPS.2-Keto-3-deoxy-D-manno-octonic acid (KDO) is a unique eight-carbon sugar commonly found in the inner core region of gram-negative bacterial lipopolysaccharides (LPS) (14), but it was considered virtually missing in vibrios (9, 10). However, the observed KDO-specific antigenicity of Vibrio cholerae LPS led to the finding of phosphorylated KDO by gas chromatography-mass spectrometry (GC-MS) (1). By using a thiobarbituric acid (TBA) assay alone, KDO has been recognized in thiobarbiturate-negative LPS prepared from Pseudomonas spp., Bacteroides spp., and Aeromonas spp. after dephosphorylation (4). No conclusive evidence for the general existence of KDO in Vibrio parahaemolyticus LPS has been reported. The purpose of this study was to demonstrate the existence of KDO in V. parahaemolyticus LPS by GC-MS and chemical methods.Six strains of V. parahaemolyticus were studied. Their serotypes and Kanagawa phenomena were as follows: 38C1, 04:K11 and K+; P7, 04:K4 and K+; P26, 02:K3 and K+; 38C6, 03:K30 and K-; P68, 04:K34 and K-; and P6, 02:K30 and K-. Kanagawa-positive (K+) strains are able to lyse red blood cells on blood agar medium containing 7% NaCl (16). Virtually all pathogenic V. parahaemolyticus strains are Kanagawa positive (17). Three strains of either K+ or K-V. parahaemolyticus were used for this study to determine whether there is any correlation between the presence of KDO and Kanagawa phenomenon. Bacteria were grown in PPBE broth (1% proteose peptone, 0.2% beef extract, 2.5% NaCl) at 35°C. Cells were harvested at an optical density at 660 nm of 1.0 to 1.2 and then lyophilized. LPS was extracted by the phenol-water method (18) and purified by repeated high-speed centrifugation (10,000 x g, 30 min) and ultracentrifugation (105,000 x g, 3 h) (7). LPS was dephosphorylated in 48% HF at 4°C for 72 h in a polyethylene centricone. HF was removed either by evaporation under a vacuum (13) or by dialysis (12). Mild acid hydrolysis with 5% acetic acid at 100°C for 5 h was used to break the glycosidic linkage between polysaccharide and lipid A in control (untreated) and dephosphorylated LPS. KDO in degraded polysaccharide was derivatized by the procedure described by Waeghe et al. (15) tions. Degraded polysaccharide underwent the following derivatization steps: (i) prereduction of the keto group by NaBD4; (ii) methylation of the hydroxyl groups of sugars by methylsulfinyl carbanion (CH3 * SO -CH2-, Na+) and CH3I by the Hakomori method (6) (methylsulfinyl carbanion was pr...

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus

“…A high level of phosphorus in V. parahaemolyticus LPS has also been reported by Deneke and Colwell (8). The 3-deoxyoctulosonic acid in the LPSs of V. cholerae and V. parahaemolyticus has been found to be virtually completely phosphorylated (6,16). Highly phosphorylated LPS may be critical for survival of V. parahaemolyticus in natural aquatic environments (8).…”