Takehiro Iguchi

Structural analysis of the carbohydrate backbone of Vibrioparahaemolyticus 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.

An immunochemical study of serological cross-reaction between lipopolysaccharides from Vibrio cholerae O22 and O139.

A comparative chemical and serological study of the LPS of Vibrio cholerae O139 and O22 was performed. Chemical analysis revealed that the sugar composition of the LPS of strain O22 was quite similar to that of O139 LPS. Each contained D-glucose, L-glycero-D-manno-heptose, colitose (3,6-dideoxy-L-galactose), D-fructose, D-glucosamine, D-quinovosamine and D-galacturonic acid. The O-antigenic relationship between the two strains was analysed by passive haemolysis (PH) and passive haemolysis inhibition (PHI) tests with the respective LPS being used as antigens to sensitize sheep red blood cells (SRBC) and, in the latter case, as inhibitors in a PH system that consisted of LPS-sensitized SRBC, guinea-pig complement and anti-O139 or anti-O22 antiserum, both unabsorbed and absorbed with the heterologous antigen. In the PH experiment, unabsorbed anti-O139 antiserum had haemolytic titres of 66,000 and 22,000 against O139 LPS- and O22 LPS-sensitized SRBC, respectively; unabsorbed anti-O22 antiserum had haemolytic titres of 900 and 13,000, respectively. Thus, the anti-O139 antiserum contained an antibody that reacted with a heterologous O22 antigen at a high titre (22,000) and this antibody was completely removed from anti-O139 antiserum with the O22 antigen. The anti-O22 antiserum contained an antibody that reacted with the heterologous O139 antigen at a low titre (900) and this antibody was completely removed from anti-O22 antiserum with the O139 antigen. In PHI tests O139 LPS and O22 LPS each strongly inhibited (the ID50 of LPS ranged from 0.03 to 0.14 microgram ml-1) the heterologous haemolytic systems of both O139 LPS-sensitized SRBC/anti-O22 antiserum and O22 LPS-sensitized SRBC/anti-O139 antiserm, which are substantially equivalent to the common antigen factor in the O139 LPS-sensitized SRBC/anti-O22 antiserum system and the common antigen factor in the O22 LPS-sensitized SRBC/anti-O139 antiserum system, respectively. The results indicated that the O antigen of O139 is closely related to that of O22 in an a,b-a,c type of relationship where a is common antigenic factor, b is an O139-specific antigenic factor and c is an O22-specific antigenic factor.

Occurrence of Uronic Acid in Lipopolysaccharides of Vibrionaceae

The occurrence of uronic acid as a sugar constituent of lipopolysaccharides (LPS) in Vibrionaceae was demonstrated for the first time. More than 100 strains were examined. Of five genera constituting Vibrionaceae, i.e., Vibrio, Aeromonas, Plesiomonas, Photobacterium, and Lucibacterium, the latter three contained uronic acid in LPS of all of their constituting members examined, while it was totally lacking in Aeromonas LPS so far tested. Only the members of genus Vibrio were found to be divided into uronic acid‐containing and ‐lacking groups; V. parahaemolyticus, V. alginolyticus, V. fisheri, V. costicola, Beneckea (‘Vibrio’), and V. fluvialis belonged to the former, while all four biotypes of V. cholerae regardless of their serotypes, V. vulnificus and V. anguillarum, belonged to the latter group.

Structure and serological characterization of 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid isolated from lipopolysaccharides of Vibrio parahaemolyticus O2 and O-untypable strain KX-V212

Lipopolysaccharides (LPS) of Vibrio parahaemolyticus O2 and O-untypable (OUT) strain (KX-V212) isolated from an individual patient were shown to contain 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid (NonlA), which was readily released from LPS by mild acid hydrolysis. In the present study, we investigated the chemical and serological properties of NonlA isolated from LPS of V. parahaemolyticus O2 and OUT KX-V212. GC-MS and NMR analysis identified the NonlA from LPS of O2 to be 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAcNonlA) and that from LPS of KX-V212 to be 5-acetamido-7-(N-acetyl-D-alanyl)amido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAlaNAcNonlA). In ELISA inhibition analysis, 5NAc7NAcNonlA inhibited the O2 LPS/anti-O2 antiserum system, whereas, 5NAc7NAlaNAcNonlA did not show any inhibitory activity. However, after N-deacylation of 5NAc7NAlaNAcNonlA followed by N-acetylation, the product (5NAc7NAcNonlA) inhibited the O2 LPS/anti-O2 antiserum system to the same extent as that of 5NAc7NAcNonlA obtained from O2 LPS. These results suggest that 5NAc7NAcNonlA might be related to the serological specificity of O2 LPS as one of main epitope(s) involved in O2 LPS.

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 (Weissbachs 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.

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1:Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O‐polysaccharide chain composed of an α(1 → 2)‐linked N‐(3‐deoxy‐L‐glycero‐tetronyl)‐D‐perosamine (4‐amino‐4,6‐dideoxy‐D‐manno‐pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N‐(3‐deoxy‐L‐glycero‐tetronyl)‐2‐O‐methyl‐D‐perosamine at the non‐reducing terminus of the O‐polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co‐existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O‐antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O‐forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2‐O‐methyl group at the non‐reducing terminus of the α(1 → 2)‐linked N‐(3‐deoxy‐L‐glycero‐tetronyl)‐D‐perosamine homopolymer that forms the O‐polysaccharide chain of LPS of V. cholerae O1 (Ogawa).

Lipopolysaccharides of group F, a new group of vibrios.

Abstract The sugar composition of the O-antigenic lipopolysaccharides isolated from Group F vibrios was analysed. 2-Keto-3-deoxy-octonate was totally absent from the lipopolysaccharides. As common component sugars, glucose, galactose, L-glycero-D-mannoheptose, and glucosamine were present. The Group F vibrios examined were found to be divided into two groups, designated tentatively as groups I and II, on the basis of the pattern of the sugar composition of their lipopolysaccharides. As additional sugar components, mannosamine, quinovosamine and two unidentified amino sugars, F1 and F2, were present in group I, while rhamnose, galactosamine, an unidentified amino sugar, F3, and a relatively high content of D-glycero-D-mannoheptose were found in group II.

A rapid method for sugar analysis of lipopolysaccharides of Gram-negative bacteria

Summary A simple and rapid method was developed for preparing and analysing the “degraded polysaccharide” (DPS) fraction virtually corresponding to the polysaccharide portion of lipopolysaccharides (LPS) from cells of Gram-negative bacteria without separating LPS. Salmonella typhimurium LT2 was used as a standard for establishing the method. It was successfully applied not only to S. typhimurium but also to Escherichia coli, Shigella flexneri, Yersinia enterocolitica, Plesiomonas shigelloides , O1 and non-O1 Vibrio cholerae, Vibrio fluvialis, Vibrio vulnificus and Vibrio anguillarum . The sugar composition of the samples prepared directly from the cells of each of these strains by the rapid method and that of the DPS fraction isolated from LPS prepared by Westphals standard phenol-water technique was shown to be identical. However, in the case of O1 and non-O1 V. cholerae and the other three vibrio species, 2-keto-3-deoxyoctonate (KDO) was undetectable by the conventional colour test (Weissbachs reaction) under conventional hydrolysis conditions. In spite of this limitation, the rapid method is considered to be suitable to determine the sugar composition of the polysaccharide portion of LPS of Gram-negative bacteria with chemotaxonomic studies.

Sugar Composition of the Polysaccharide Portion of Lipopolysaccharides of Vibrio fluvialis, Vibrio vulnificus, and Vibrio mimicus

A chemotaxonomic study was carried out on Vibrio fluvialis and V. vulnificus on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharides (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. Nineteen O‐serogroups of V. fluvialis were divided into 14 chemotypes while seven O‐serogroups of V. vulnificus were divided also into seven chemotypes since the polysaccharide portion of LPS of each serogroup has a different sugar composition from that of the other serogroups. Close similarities in the sugar composition of the same portion were demonstrated between serologically cross‐reacting non‐O1 group V. cholerae and V. fluvialis, and non‐O1 V. cholerae and V. mimicus.

Chemotaxonomic study of Vibrio cholerae bio-serogroup Hakata on the basis of the sugar composition of the polysaccharide portion of its lipopolysaccharides.

A chemotaxonomic study was carried out on 31 strains of non‐O1 Vibrio cholerae bio‐serogroup Hakata, isolated in Japan, which possesses the Inaba antigen C of O1 V. cholerae. On the basis of the compositional sugar pattern of the polysaccharide portion of their lipopolysaccharides, the 23 strains isolated from the environment were separated into two groups, one (20 strains) containing mannose, glucose, fructose, L‐glycero‐D‐mannoheptose, glucosamine, perosamine, quinovosamine, and an unidentified amino sugar AS, and the other (3 strains) containing two additional sugars, galactose and a trace amount of galactosamine. All of the eight strains isolated from imported seafoods belonged to the former group.