Chemical structure of the 2-keto-3-deoxyoctonate (KDO) region of the lipopolysaccharide isolated from O1 Vibrio cholerae NIH 4IR (Ogawa).

The chemical structure of the 2-keto-3-deoxyoctonate (KDO) region of the lipopolysaccharide (LPS) isolated from O1 V. cholerae NIH 41R (Ogawa) was elucidated by dephosphorylation, periodate oxidation and methylation analysis. Methylation analysis of KDO in the dephosphorylated LPS revealed the presence of 5-O-acetyl-1,2,4,6,7,8-hexa-O-methyl-3-deoxy-octitol and 2-keto-3-deoxy-heptulosonic acid was detected in the methanolysate of the periodate-oxidized and dephosphorylated LPS. These results indicated that the site of binding of KDO to the core oligosaccharide is position C5 as in enteric gram-negative bacterial LPS, while only one molecule of the KDO residue carrying phosphate on position C4 is present in the inner core region of the LPS in contrast to enteric gram-negative bacterial LPS in which one molecule of KDO carrying KDO or KDO2----4KDO disaccharide instead of the phosphate group at position C4 is present in its main chain.     

Characterization of gonidial zone of Cycas revoluta coralloid roots by means of microelectrodes

Abstract Structural analysis of the 2-keto-3-deoxyoctonate region of lipopolysaccharide (LPS) isolated from Porphyromonas (Bacteroides) gingivalis was carried out. The substitution of the polysaccharide portion on the KDO was determined by gas chromatography/mass spectrometry of the product obtained by sequential derivatization of the LPS, including dephosphorylation, permethylation, carboxyl reduction, partial hydrolysis, carbonyl reduction, complete hydrolysis and O -acetylation. It was revealed that the KDO carries the polysaccharide on its position C5 and is phosphorylated on either position C7 or C8, although its exact position is not determined. The structure of the KDO region of P. gingivalis LPS in Gram-negative bacterial LPS had not hitherto been elucidated.     

Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide.

A phosphorylated 3-deoxy-manno-octulosonic acid (KDO) was released from the lipopolysaccharide (LPS) of the deep rough mutant (Rb+169) of Haemophilus influenzae by acid hydrolysis. Both phosphorylated and dephosphorylated KDO, produced by treatment with alkaline phosphatase, were identified by gas chromatography-mass spectrometry after trimethylsilylation. This technique provides a rapid and reliable method for the identification of phosphorylated KDO in LPS.     

Analysis of the 2-keto-3-deoxyoctonate (KDO) region of lipopolysaccharides isolated from non-01 Vibrio cholerae 05R

Phosphorylated 2-keto-3-deoxyoctonate (KDO) has been detected in the strong-acid hydrolysates of lipopolysaccharides (LPS) of family Vibrionaceae including Vibrio cholerae. Structural analysis of LPS isolated from a rough mutant of non-01 V. cholerae 05 by dephosphorylation, periodate oxidation and methylation analysis revealed that the inner core region of the LPS molecule contains only one mole of KDO in contrast to enteric Gram-negative bacterial LPS, and that the phosphate group on the KDO molecule resides in the C4 position, while the site of binding of KDO to heptose, a constituent of the distal part of the inner core region, is the C5 position as in the enteric bacterial LPS.     

Chemotypes of Fusobacterium nucleatum lipopolysaccharides

Lipopolysaccharides (LPS) were isolated from 20 strains of Fusobacterium nucleatum and examined by paper chromatography, gas liquid chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-dexoxy-octonate (KDO). The LPS had in common glucosamine, L-glycero-D-manno-heptose, glucose and KDO. The KDO content was low. Galatose, rhamnose and D-glycero-D-manno-heptose were found in some strains. Based on the sugar composition of the LPS, the F. nucleatum strains could be classified into six chemotypes.     

Characterization of the carbohydrate backbone of Vibrio parahaemolyticus O6 lipopolysaccharides

Structural characterization studies have been carried out on the carbohydrate backbone of Vibrio parahaemolyticus serotype O6 lipopolysaccharides (LPS). The carbohydrate backbone isolated from O6 LPS by sequential derivatization, that is, dephosphorylation, O-deacylation, pyridylamination, N-deacylation and N-acetylation, is a nonasaccharide consisting of 3 mol of D-glucosamine (GlcN) (of which one is pyridylaminated), 2 mol of L-glycero-D-manno-heptose (Hep), and 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA) and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Structural analyses by nuclear magnetic resonance spectroscopy and fast-atom bombardment mass spectrometry demonstrated that the carbohydrate backbone is β-Galp-(1→2)-α-Hepp-(1→3)-α-Hepp-(1→5)-α-Kdop-(2→6)-β-GlcpNAc-(1→6)-GlcNAc-PA, in which the 3-substituted α-Hepp is further substituted by β-GlcpNAc-(1→4)-β-Glcp at position 4 and by β-GlcpA at position 2. In native O6 LPS, an additional 1 mol of D-galacturonic acid, which is liberated by dephosphorylation in hydrofluoric acid, is present at an unknown position. A previous study by the present authors reported that, of 13 O-serotype LPS of V. parahaemolyticus, the only LPS from which Kdo was detected was from O6 LPS after mild acid hydrolysis. In the present study, we have demonstrated that only 1 mol of Kdo is present at the lipid A proximal position, a component which is common to the LPS in all serotypes of the bacterium, and that there is no additional Kdo in the carbohydrate backbone of O6 LPS. ELISA and ELISA inhibition analysis using antisera against O6 and Salmonella enterica Minnesota R595 and LPS of both strains further revealed that Kdo is not involved as an antigenic determinant of O6 LPS.     

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient

Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient. Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V. parahaemolyticus serotype O2. A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep). Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone. Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below. The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions. Both similarity and differences are observed between the LPS of V. parahaemolyticus serotype O2 and strain KX-V212. [carbohydrate structure: see text]     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Occurrence of 2-keto-3-deoxyoctonate (KDO) and KDO phosphate in lipopolysaccharides ofBacteriodes species

Occurrence of 2-keto-3-deoxyoctonate (KDO) in lipopolysaccharides (LPS) of genusBacteroides (some strains have recently been reclassified asPorphyromonas orPrevotella) was examined. Strong-acid treatment of LPS isolated fromBacteroides fragilis, Bacteroides (Porphyromonas) gingivalis andBacteroides intermedius, (Prevotella intermedia) released periodate/thiobarbituric acid reaction-positive substances that were not detectable under conventional hydrolysis conditions. These substances were demonstrated to be KDO phosphate by high voltage paper electrophoresis before and after alkaline phosphatase treatment. KDO phosphate was also identified in these LPS by gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. KDO was identified as well in both mild and strong-acid hydrolysates of LPS isolated fromBacteriodes melaninogenicus (Prevotella melaninogenica). Neither KDO nor KDO phosphate was detectable in LPS ofBacteriodes asaccharolyticus (Porphyromonas asaccharolytica) even after the strong-acid treatment of LPS. These findings indicate that there are possible structural variations in the inner core region ofBacteroides LPS.     

Chemotypes of Veillonella lipopolysaccharides

Lipopolysaccharides (LPS) were isolated by phenol-water extraction from 34 strains of Veillonella, and examined by paper chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-deoxy-octonate (KDO). Several preparations were also examined for neutral sugars by gas liquid chromatography. The LPS had in common glucosamine, galactosamine, L-glycero-D-manno-heptose glucose and KDO. Most LPS contained galactose, and a few rhamnose. D-glycero-D-manno-heptose was found in LPS from one of the strains. Based on the sugar composition of the LPS, the Veillonella strains could be classified into four chemotypes.     

Selective detection of 3-deoxymannooctulosonic acid in intact lipopolysaccharides by spin-echo 13C NMR

The 3-deoxy-D-mannooctulosonic acid (KDO) region of lipopolysaccharides (LPS) from the heptoseless mutant Salmonella minnesota R595 and inner core and heptoseless mutants derived from Escherichia coli K12 was studied by 13C NMR spectroscopy. A spin-echo spectral editing technique was employed for the selective detection of the quaternary anomeric carbon of ketosidically linked KDO. Only two quaternary carbon resonances attributable to KDO were detected in the anomeric carbon spectral region of each LPS from heptoseless mutants E. coli D31m4 (99.7 and 100.8 ppm) and S. minnesota R595 (100.0 and 100.9 ppm). Integrated signal intensities from fully relaxed normal 13C spectra showed that equivalent molar quantities of KDO and glucosamine (i.e. 2 mol of each) were present in each of these samples. Similarly, only two KDO anomeric carbon resonances were detected in the LPS from the inner core mutants E. coli D21f1 (100.8 and 101.2 ppm) and E. coli D21e7 (100.8 and 101.2 ppm). These data confirm the presence of a KDO disaccharide structure rather than a trisaccharide as determined by others using thiobarbituric acid-based assays. The LPS of E. coli D21 (complete inner core oligosaccharide) exhibited four quaternary anomeric carbon resonances (99.4, 100.7, 101.8, and 102.7 ppm). The unequal intensities of these resonances, however, demonstrated that significant heterogeneity exists with respect to KDO substitution in this LPS. A third KDO moiety present in substoichiometric amounts could be consistent with this observation. However, this possibility could not be distinguished from other modes of substitutional heterogeneity involving only 2 KDO residues.     

Ultrastructure and chemical composition of lipopolysaccharide extracted from Leptospira interrogans serovar copenhageni

Lipopolysaccharide (LPS) from Leptospira interrogans serovar copenhageni was prepared from the aqueous phase of a phenol/water extract. Electron microscopic examination of negatively stained LPS showed a mixture of ribbon-like, round and ring structures. Carbohydrate analysis of the preparations revealed pentoses, hexoses, heptoses, hexosamines, and a 2-keto-3-deoxyonic acid which was chromatographically different from authentic 2-keto-3-deoxyoctonic acid (KDO). The major fatty acids of the LPS were hydroxylauric, palmitic and oleic acids. Although the leptospiral LPS preparations did not contain KDO or hydroxymyristic acid, they were otherwise morphologically and chemically similar to the LPS of other Gram-negative bacteria.     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram-negative bacteria

A procedure is described to determine 2-keto-3-deoxyoctonate (KDO) present in lipopolysaccharide (LPS) of gram-negative bacteria. The method involves the treatment of LPS with 0.2 n H₂SO₄ at 100°C for 30 min to release KDO, followed by its reaction with periodic acid, sodium arsenite, and thiobarbituric acid. The red chromophore thus formed is kept in solution at room temperature by adding dimethylsulfoxide to the reaction mixture. The final color is stable for days at room temperature and facilitates accurate determination of KDO in microgram quantities. KDO contents of cell surface antigens and glycolipids from gram-negative bacteria are presented as illustrations of the accuracy and sensitivity of the assay.     

Production and characterization of KDO-specific monoclonal antibodies recognizing lipopolysaccharides from heptoseless mutants of Salmonella

Abstract Hybrid cell lines producing monoclonal antibodies with specificity for the lipopolysaccharide (LPS) from the deep rough mutant Salmonella minnesota R595 have been established. Spleen cells from BALB/c mice immunized with live R595 bacteria were fused with Sp 2/0 myeloma cells and three hybridomas producing antibodies specific for heptoseless LPS from Salmonella were selected. All three monoclonal antibodies were shown to bind only to heptoseless, but 3-deoxy- d -manno-octulosonic acid (KDO) containing LPS when tested in enzyme-linked immunosorbent assay (ELISA) against a set of structurally defined LPS and lipid A from Salmonella, Shigella and Escherichia coli . Synthetic KDO was an efficient inhibitor of the antibody-R595 LPS interaction defining that KDO is in an immunodeterminant position interacting with the monoclonal antibodies.     

Occurrence of O-phosphorylated 2-keto-3-deoxyoctonate in the lipopolysaccharide of Bacteroides gingivalis

Abstract Periodate-thiobarbituric acid reaction-positive substances were found in the strong acid hydrolysates of the lipopolysaccharide (LPS) from Bacteroides gingivalis 381. They were not identical to 2-keto-3-deoxyoctonate (KDO) in high-voltage paper electrophoresis (HVPE), their electrophoretic mobilities relative to KDO being 1.54 and 1.80, respectively. Alkaline phosphatase treatment and HVPE demonstrated that they are some kind of O-phosphorylated derivatives of KDO; in particular, the slow-moving component is identical, at least in HVPE, to 5- O -phosphoryl-KDO isolated from the strong acid hydrolysates of Bordetella pertussis (phase I) LPS.     

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.     

Methylation analysis of the heptose/3-deoxy-D-manno-2-octulosonic acid region (inner core) of the lipopolysaccharide from Salmonella minnesota rough mutants

A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy-D-manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd2P-, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di-O-acetyl and 2,4,6-tri-O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd2P- LPS, the hydrolysis step involved 0.1M trifluoroacetic acid at 100 degrees for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:02 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave 5-O-acetyl-3-deoxy-1,7,8-tri-O-methyl-2,4,6-tri-O-trideuteriomethyl++ +-D- glycero-D-talo/galacto-octitol and 1,5-di-O-acetyl-2,3,4,6,7-penta-O-methyl-L-glycero-D-manno-heptitol++ +. These results prove the presence of a (2----4)-linked KDO disaccharide in Re LPS and show that the core region of Rd2P- LPS contains a terminal alpha-L-glycero-D-manno-heptopyranosyl group and a non-substituted, a 4-O-, and a 4,5-di-O-substituted pyranosidic KDO residue in the molar ratios 1:1:0.2:1.     

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

A partition chromatographic procedure utilizing a cationic exchange resin column in the Li⁺ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of R_e and R_dP^? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R_e or R_dP^? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H⁺ form) released more than 80% of the KDO residues within 15 min. The heptose of the R_dP^? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R_e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R_dP^? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.     

Hydrolytic release, and identification by g.l.c.-m.s., of 3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharides isolated from bacteria of the Vibrionaceae

The identification of the peracetylated methyl glycosides of 3-deoxy-D-manno-2-octulosonic acid (KDO) methyl esters was achieved by g.l.c.-m.s. These peracetylated methyl glycoside methyl esters were obtained from fully acetylated lipopolysaccharides and core oligosaccharides of representative strains of the Vibrionaceae family by the following sequence of mild reactions: acetolysis, methanolysis, and acetylation. KDO was shown to be present in all of the lipopolysaccharides (LPS), a result in direct contrast to the generally accepted view of the absence of this compound in LPS from this family of bacteria.