The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group G (o 13,22) lipopolysaccharides

1. Lipopolysaccharides have been isolated from ;smooth' (S) strains of Salmonella friedenau and Salmonella poona by the phenol-water method and purified in the preparative ultracentrifuge. 2. These lipopolysaccharides are serologically indistinguishable and on partial acid hydrolysis the same series of oligosaccharides was obtained in each instance. 3. The results of quantitative micro-analysis, borohydride reduction, periodate oxidation, Morgan-Elson reactions and enzymic hydrolysis with beta-galactosidase on the isolated oligosaccharides indicate that the O-specific side chains of these lipopolysaccharides have a repeating pentasaccharide unit that is beta-d-galactosyl-(1-->3)-N-acetylgalactosaminyl-(1-->3)-N-acetylgalactosaminyl-(1-->4)-l-fucose with a d-glucose residue bound at an undetermined point on this structure. 4. Two oligosaccharides, a glucosyl-galactose and an N-acetylglucosaminylglucose, have also been isolated and these seem to be identical with oligosaccharides obtained from ;rough' (R) Salmonella lipopolysaccharides. These findings are in accordance with the view that Salmonella S-lipopolysaccharides have a core that consists of R-lipopolysaccharide.     

The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group N (o 30) lipopolysaccharides

1. The lipopolysaccharides isolated from ;smooth' (S) strains of Salmonella godesberg and Salmonella urbana by the phenol-water method were purified in the ultracentrifuge. 2. These lipopolysaccharides have the same O-antigenic structure and on partial hydrolysis the same series of oligosaccharides was obtained in each instance. 3. The results of quantitative microanalysis, borohydride reduction, periodate oxidation, Morgan-Elson reactions and enzymic hydrolysis with alpha- and beta-glucosidases on the isolated oligosaccharides indicated that the O-specific side chains of these S-lipopolysaccharides have a repeating tetrasaccharide unit that is beta-d-glucosyl-(1-->3)-N-acetylgalactosaminyl-(1-->4)-l-fucose with a further glucose residue bound at the 4-position on the N-acetylgalactosamine. 4. Another oligosaccharide, a glucosylgalactose, has also been isolated and is indistinguishable from an oligosaccharide isolated from Salmonella R-lipopolysaccharides. These findings provide further evidence supporting the view that all Salmonella S-lipopolysaccharides have a core consisting of R-lipopolysaccharide.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.     

Studies on the structure of lipopolysaccharides of Salmonella minnesotta and Salmonella typhimurium R strains

1. The composition of the lipopolysaccharides and the corresponding lipid-free polysaccharides from four R-mutants of Salmonella has been studied. All the lipopolysaccharides, from RI and RII serotypes contained d-glucose, d-galactose, heptose, N-acetylglucosamine and 3-deoxy-2-oxo-octonate. The polysaccharide obtained from the RII lipopolysaccharides also contained all these sugars. The polysaccharides from RI lipopolysaccharides lacked N-acetylglucosamine. 2. From partial hydrolysates of the lipopolysaccharides, a number of oligosaccharides have been isolated and partially characterized. Oligosaccharides containing N-acetylglucosamine or glucosamine were obtained only from RII lipopolysaccharides. Several oligosaccharides composed of glucose and galactose were common to RI and RII preparations. 3. A structural unit, based on the oligosaccharides found, is proposed for the RII lipopolysaccharide. It contains the sequence: alpha-N-acetylglucosaminyl- alpha-glucosyl-alpha-galactosyl-glucosyl.... A second alpha-galactosyl residue is bound to position 6 of the last glucosyl group. The complete unit is believed to to be attached to a polyheptose phosphate backbone in the RII antigen. 4. The RI lipopolysaccharide of Salmonella minnesota contains an analogous structure lacking the terminal N-acetylglucosamine residue. 5. A basal structure common to the lipopolysaccharides of several Salmonella species is proposed.     

Core oligosaccharides of Plesiomonas shigelloides O54:H2 (strain CNCTC 113/92): structural and serological analysis of the lipopolysaccharide core region, the O-antigen biological repeating unit, and the linkage between them.

The structure of the core oligosaccharide moiety of the lipopolysaccharide (LPS) of Plesiomonas shigelloides O54 (strain CNCTC 113/92) has been investigated by (1)H and (13)C NMR, fast atom bombardment mass spectrometry (MS)/MS, matrix-assisted laser-desorption/ionization time-of-flight MS, monosaccharide and methylation analysis, and immunological methods. It was concluded that the main core oligosaccharide of this strain is composed of a decasaccharide with the following structure: (see text) in which l-alpha-D-Hepp is l-glycero-alpha-D-manno-heptopyranose. The nonasaccharide variant of the core oligosaccharide ( approximately 10%), devoid of beta-D-Glcp substituting the alpha-D-GlcpN at C-6, was also identified. The core oligosaccharide substituted at C-4 of the outer core beta-D-Glcp residue with the single O-polysaccharide repeating unit was also isolated yielding a hexadecasaccharide structure. The determination of the monosaccharides involved in the linkage between the O-specific polysaccharide part and the core, as well as the presence of -->3)-D-beta-D-Hepp-(1--> instead of -->3,4)-D-beta-D-Hepp-(1--> in the repeating unit, revealed the structure of the biological repeating unit of the O-antigen. The core oligosaccharides are not substituted by phosphate residues and represent novel core type of bacterial LPS that is characteristic for the Plesiomonas shigelloides serotype O54. Serological screening of 69 different O-serotypes of P. shigelloides suggests that epitopes similar to the core oligosaccharide of serotype O54 (strain CNCTC 113/92) might also be present in the core region of the serotypes O24 (strain CNCTC 92/89), O37 (strain CNCTC 39/89) and O96 (strain CNCTC 5133) LPS.     

Smith degradation of the O-antigenic polysaccharide of Salmonella Dakar: structural studies of the products

Two different oligosaccharides were obtained from the Smith degradation of the O-polysaccharide isolated from the lipopolysaccharide of Salmonella Dakar. The structures of these oligosaccharides were investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. The following structures of these products were determined: alpha-D-GalpNAc-(1-->4)-alpha-D-Quip3NAc-(1-->3)-alpha-L-Rhap-(1-->2)-threitol and [FORMULA: SEE TEXT] where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. The reaction products confirmed the structure of the repeating unit of the Salmonella Dakar O-polysaccharide reported previously [Kumirska, J.; Szafranek, J.; Czerwicka, M.; Paszkiewicz, M.; Dziadziuszko, H.; Kunikowska, D.; Stepnowski, P. Carbohydr. Res. 2007,342, 2138-2143].     

The structure of the phosphorylated carbohydrate backbone of the lipopolysaccharide of the phytopathogen bacterium Pseudomonas tolaasii

A novel core-lipid A backbone oligosaccharide was isolated and identified from the lipopolysaccharide fraction of the mushrooms pathogen bacterium Pseudomonas tolaasii. The oligosaccharide was obtained by alkaline treatment of the lipopolysaccharide fraction. Since the repeating unit of the O-antigen contained one residue of -->4)-alpha-l-GulpNAcAN, the hydrolysis was accompanied by beta-elimination on this residue and following depolymerization, producing a mixture of oligosaccharides. The complete structural elucidation showed the presence of a single core glycoform and was achieved by chemical analysis and by (1)H, (31)P, and (13)C NMR spectroscopy applying various 1D and 2D experiments. [structure: see text]. All sugars are alpha-d-pyranoses, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid, P is phosphate. QuiN and DeltaGulNA are present in nonstoichiometric amount.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O28

An O-polysaccharide and oligosaccharides were isolated by GPC following mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O28. The O-polysaccharide was studied by sugar and methylation analyses, ¹H and ¹³C NMR spectroscopy, including 2D ROESY and H-detected ¹H,¹³C HSQC and HMBC experiments, and the following structure of the branched pentasaccharide repeating unit was established:This structure was confirmed by ESI MS of the isolated tridecasaccharide consisting of the lipopolysaccharide core and one O-polysaccharide repeat. The ESI mass spectrum also enabled inferring the composition of the core oligosaccharide.     

Structure of the sialic acid-containing O-specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide.

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 degrees C or 0.1 M NaOH-AcOH, pH 4.5, 5 h, 100 degrees C), the O-specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O-specific polysaccharide was shown to be the following trisaccharide: -->4)-alpha-Neup5Ac(2-->3)-L-alpha-FucpNAc(1-->3)-D-beta-Glc pNAc(1--> The polysaccharide O-chain was substituted with a single molar equivalent of O-acetyl group, distributed between the Neu5Ac O-9 and O-7 positions, in an approximate ratio of 7 : 3.     

Characterisation of the Salmonella O:8 antigen in the O-chain of the lipopolysaccharide produced by Salmonella virginia O:8.

The antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella virginia (O:8), analyzed by methylation, partial acid hydrolysis, and one- and two-dimensional nuclear magnetic resonance methods, was found to be a polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, D-abequose, and O-acetyl (2:1:1:1:1.3) and having the following structure: [formula; see text] The disaccharide structure alpha-D-Abep-(1----3)-L-Rhap was identified as the Salmonella O:8 antigenic factor epitope, since the removal of alpha-D-Abep residues from the O-polysaccharide left a residual tetrasaccharide repeating unit backbone that did not show reaction with Salmonella type O:8 factor antiserum.     

Structure of the O-specific polysaccharide of Proteus vulgaris O45 containing 3-acetamido-3,6-dideoxy-D-galactose

An O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O45 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established:-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Fucp3NAc4Ac-(1-->where Fuc3NAc4Ac is 3-acetamido-4-O-acetyl-3,6-dideoxygalactose. A cross-reactivity of anti-P. vulgaris O45 serum was observed with several other Proteus lipopolysaccharides, which contains Fuc3N derivatives.     

The occurrence of glycine in bacterial lipopolysaccharides

Abstract The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [¹⁴C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100°C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.     

rfaP mutants of Salmonella typhimurium

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.     

Serotyping of clinical isolates belonging to Proteus mirabilis serogroup O36 and structural elucidation of the O36-antigen polysaccharide

The O-specific polysaccharide (OPS) isolated from the lipopolysaccharide of Proteus mirabilis O36 was found to have a pentasaccharide repeating unit of the following structure: -->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAc6Ac-(1-->4)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. The structure is unique among Proteus OPS, which is in agreement with the classification of this strain into a separate Proteus O-serogroup. Remarkably, the P. mirabilis O36-polysaccharide has the same structure as the OPS of Escherichia coli O153, except that the latter is devoid of O-acetyl groups. The cross-reaction of anti-O36 antibodies with the O-part of E. coli O153 lipopolysaccharide is observed. In the present study, two steps of serotyping Proteus strains are proposed: screening of dry mass with enzyme-linked immunosorbent assay and immunoblot with the crude lipopolysaccharides. This method allowed serotyping of 99 P. mirabilis strains infecting the human urinary tract. Three strains were classified into serogroup O36. The migration pattern of these lipopolysaccharides fraction with long O-specific PSs was similar to the standard laboratory P. mirabilis O36 (Prk 62/57) lipopolysaccharide. The relatively low number of clinical strains belonging to serogroup O36 did not correspond to the presence of anti-P. mirabilis O36 antibodies in the blood donors' sera. Twenty-five percent of tested sera contained a statistically significant elevated level of antibodies reacting with thermostable surface antigens of P. mirabilis O36. The presence and amount of antibodies correlated with Thr399Ile TLR4 polymorphism types (P=0.044).     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established: -->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Galp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-GlcpNAc-(1-->.     

Structure of the O-specific polysaccharide of the lipopolysaccharide of Azospirillum brasilense Sp245

An O-specific polysaccharide was isolated from the lipopolysaccharide of a plant-growth-promoting bacterium Azospirillum brasilense Sp245 and studied by sugar analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY. The polysaccharide was found to be a new rhamnan with a pentasaccharide repeating unit having the following structure:-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->     

Serological characterisation of anti-endotoxin sera directed against the conjugates of oligosaccharide core of Escherichia coli type R1, R2, R3, J5 and Salmonella Ra with tetanus toxoid

Abstract The covalent conjugates of oligosaccharide core: Escherichia coli type R1, R2, R3, J5 and Salmonella Ra with tetanus toxoid have been prepared using reaction of reductive amination. The neoglycoconjugates were good immunogens in rabbits yielding a high level of anti-lipopolysaccharide antibodies of IgG class. The antibodies were used to examine the possibility of their reactions with smooth lipopolysaccharides. We have found that all antisera were able to react with the lipopolysaccharide molecules of identical or related core type, possessing core oligosaccharides substituted with O-specific chains. These reactions were shown in both the ELISA assay and the immunoblotting test.     

Structure of the O-antigen of the main lipopolysaccharide isolated from Sinorhizobium fredii SMH12

The lipopolysaccharide of Sinorhizobium fredii SMH12, a wide-range host bacterium isolated from nodulated soybean plants growing in Vietnam, has been studied. Isolation of lipopolysaccharide by the phenol-water method leads to a mixture of two polysaccharides; polyacrylamide gel electrophoresis indicates that both are possibly lipopolysaccharides. The structures of the O-antigen of the main lipopolysaccharide and its deacetylated form are determined by sugar and methylation analysis, partial hydrolysis, lithium degradation, ESI-MS/MS, and NMR studies. Here we show that the fast-growing S. fredii SMH12 produces a lipopolysaccharide whose O-antigen has a repeating unit consisting of the trisaccharide -->4)-alpha-D-Gal pA-(1-->3)-2-O-Ac-alpha-L-Rha p-(1-->3)-2-O-Ac-alpha-D-Man p-(1-->. The position O-6 of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in an approximate ratio 1:1:2. The tandem mass spectrometry studies rule out both an alternating and a random distribution of methyl groups and suggest the existence of zones in the polysaccharide rich in methyl groups interspersed with zones without methyl groups.     

Structural analysis of the O-antigen polysaccharide from the Shiga toxin-producing Escherichia coli O172.

The structure of the O-antigen polysaccharide from Escherichia coli O172 has been determined. In combination with sugar analysis, NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by mass spectrometry and two-dimensional NMR techniques. An O-acetyl group was present as 0.7 equivalent per repeating unit. Treatment of the O-deacetylated polysaccharide with aqueous 48% hydrofluoric acid rendered cleavage of the phosphodiester in the backbone of the polymer and the pentasaccharide isolated after gel permeation chromatography was structurally characterized. Subsequent NMR experiments on polymeric materials revealed the structure of the repeating unit of the O-polysaccharide from E. coli O172 as:-->P-4)-alpha-D-Glcp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D- GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->4)-alpha-D-Glcp6Ac-(1-->