The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.     

Structural studies of the O-specific polysaccharide from Serratia marcescens N.C.T.C. 1377

The putative O-specific polysaccharide of Serratia marcescens N.C.T.C. 1377 is a partially acetylated glucorhamnan. By means of 1H- and 12C-n.m.r. spectroscopy, methylation analysis, and periodate oxidation, it was shown that the polymer has a disaccharide repeating-unit for which the following structure is proposed: leads to 4)-alpha-D-Glcp-(1 leads to 3)-beta-L-Rhap-(1-leads. O-Acetyl groups are probably located at C-2 of the rhamnopyranosyl residues. Except for the extent of O-acetylation, the polysaccharide is identical with the corresponding product from S. marcescens Bizio (A.T.C.C. 264), for which a different structure has previously been proposed.     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of Shigella dysenteriae types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of nonstoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, the position of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by 13C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner. [Formula: see text].     

Synthesis of cytidine diphosphate-3,6-dideoxyhexoses--donors of glycosyl residues in the biosynthesis of O-specific polysaccharides of Salmonella of serogroups A, B and C

Interaction of lithium alcoholates of 2,4-di-O-benzoates of paratose and abequose with tetrabenzyl pyrophosphate gave alpha-phosphates of the 3,6-dideoxyhexoses, further converted into the corresponding cytidine-5'-diphosphate derivatives. These synthetic nucleotides were shown to participate in the biosynthesis of the O-specific polysaccharides for Salmonella typhimurium and S. nitra.     

Acetylation of O-specific lipopolysaccharides from Shigella flexneri 3a and 2a occurs in Escherichia coli K-12 carrying cloned S. flexneri 3a and 2a rfb genes.

Most of the Shigella flexneri O-specific serotypes result from O-acetyl and/or glucosyl groups added to a common O-repeating unit of the lipopolysaccharide (LPS) molecule. The genes involved in acetylation and/or glucosylation of S. flexneri LPS are physically located on lysogenic bacteriophages, whereas the rfb cluster contains the biosynthesis genes for the common O-repeating unit (D.A.R. Simmons and E. Romanowska, J. Med. Microbiol. 23:289-302, 1987). Using a cosmid cloning strategy, we have cloned the rfb regions from S. flexneri 3a and 2a. Escherichia coli K-12 containing plasmids pYS1-5 (derived from S. flexneri 3a) and pEY5 (derived from S. flexneri 2a) expressed O-specific LPS which reacted immunologically with S. flexneri polyvalent O antiserum. However, O-specific LPS expressed in E. coli K-12 also reacted with group 6 antiserum, indicating the presence of O-acetyl groups attached to one of the rhamnose components of the O-repeating unit. This was confirmed by measuring the amounts of acetate released from purified LPS samples and also by the chemical removal of O-acetyl groups, which abolished group 6 reactivity. The O-acetylation phenotype was absent in an E. coli strain with an sbcB-his-rfb chromosomal deletion and could be restored upon conjugation of F' 129, which carries sequences corresponding to a portion of the deleted region. Our data demonstrate that E. coli K-12 strains possess a novel locus which directs the O acetylation of LPS and is located in the sbcB-rfb region of the chromosomal map.     

Structure of the O-specific polysaccharide chain of the Yersinia pseudotuberculosis lipopolysaccharide (serovar II C)]

An O-specific polysaccharide has been isolated on mild acid hydrolysis of lipopolysaccharide from Yersinia pseudotuberculosis serovar IIc and shown to consist of abequose, D-mannose and 2-acetamido-2-deoxy-D-galactose residues in the ratio 0.8:3:1. From the results of acid hydrolysis, 13C NMR, methylation and periodate oxidation studies the structure of the repeating unit of the O-specific polysaccharide is deduced as follows: (formula; see text)     

Incorporation of paratose residue into Salmonella O-specific polysaccharides using enzymatic reactions]

The block mechanism of O-specific polysaccharides biosynthesis was demonstrated for Salmonella nitra (serogroup A) and S. haifa (serogroup B). Due to the moderate specificity of glycosyl transferases from S. nitra, S. typhimurium, S. haifa and S. kentucky (serogroup C3) towards the 3,6-dideoxyhexose structure a paratose residue can be incorporated into the polysaccharide chain instead of an abequose residue, and vice versa.     

The structure of the linkage between the O-specific polysaccharide and the core region of the lipopolysaccharide from Salmonella enterica serovar Typhimurium revisited.

Salmonella enterica sv. Typhimurium strain 1135 possesses smooth(S)-form lipopolysaccharide (LPS). Although the structures of the core region and the O-specific polysaccharide were investigated intensively between the 1960s and the 1980s, the structure of the linkage region between the O-chain and the core was not elucidated unequivocally. By using modern MS and high-field NMR spectroscopy for analysis of the isolated carbohydrate backbone of the LPS, it has been shown that it is a beta-D-Galp residue that links the first repeating unit of the O-specific polysaccharide to O-4 of the last D-Glcp residue of the core region. Interestingly, this particular D-Galp residue is alpha-linked in all following repeating units. The data are discussed with regard to the ligation of O-specific polysaccharide and core region during LPS biosynthesis.     

Structure of an acidic O-specific polysaccharide from marine bacterium Shewanella fidelis KMM 3582T containing Nepsilon-[(S)-1-carboxyethyl]-Nalpha-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of the marine bacterium Shewanella fidelis type strain KMM 3582T and studied by sugar analysis along with 1H and 13C NMR spectroscopy including one-dimensional NOE in difference mode and two-dimensional experiments. The polysaccharide was found to consist of linear tetrasaccharide repeating units containing Nepsilon-[(S)-1-carboxyethyl]-Nalpha-(D-galacturonoyl)-L-lysine and having the following structure: [See text.] The amide of D-galacturonic acid with Nepsilon-[(S)-1-carboxyethyl]-L-lysine ('alaninolysine', 2S,8S-AlaLys) was found for the first time in nature as a component of the O-specific polysaccharide of Providencia rustigianii O14 (Carbohydr. Res. 2003, 338, 1009-1016).     

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens IMV 2366 (biovar C)

The O-specific polysaccharide of P. fluorescens IMV 2366 was studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D gsCOSY, TOCSY, gsNOESY, H-detected 1H,(13)C gsHSQC, HMQC-TOCSY, and gsHMBC experiments. The polysaccharide contains L-rhamnose, 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) and 3-acylamido-3,6-dideoxy-D-glucose (D-Qui3NAcyl, where Acyl is 3-hydroxy-2,3-dimethyl-5-oxoprolyl). The structure 1 of the polysaccharide was found to be similar to the structure 2 of a 6-deoxy-L-talose (L-6dTal)-containing O-specific polysaccharide of a non-classified P. fluorescens strain, 361, studied earlier [Khomenko, V. A.; Naberezhnykh, G. A.; Isakov, V. V.; Solov'eva, T. F.; Ovodov, Y. S.; Knirel, Y. A.; Vinogradov, E. V. Bioorg. Khim. 1986, 12, 1641-1648; Naberezhnykh, G. A.; Khomenko, V. A.; Isakov, V. V., El'kin, Y. N.; Solov'eva, T. F.; Ovodov, Y. S. Bioorg. Khim. 1987, 13, 1428-1429]. --> 2)-beta-D-Quip3NAcyl-(1 --> 3)-alpha-L-Rhap-(1 --> 3)-alpha-D-FucpNAc-(1 --> 1. --> 4)-beta-D-Quip3NAcyl-(1 --> 3)-alpha-L-6dTalp4Ac-(1 --> 3)-alpha-D-FucpNAc-(1 -->2.     

Structures of the O1B and O1C lipopolysaccharide antigens of Escherichia coli.

The O-specific moieties of the O1B antigen (lipopolysaccharide) from Escherichia coli O1B:K1 and the O1C antigen from E. coli O1C:K- both consist of L-rhamnose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine in a molar ratio of 2:1:1:1. By using fragmentation procedures, methylation analysis, and one- and two-dimensional nuclear magnetic resonance spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In the O1B polysaccharide X is 2, and in the O1C polysaccharide X is 3. With the recently published structure of the O1A polysaccharides (B. Jann, A. S. Shashkov, D. S. Gupta, S. M. Panasenko, and K. Jann, Carbohydr. Polym. 18:51-57 1992), three related O1 antigens are now known. Their common (O1-specific) epitope is suggested to be the side-chain N-acetyl-D-mannosamine residue.     

Structural characterization of the O-specific polysaccharide from the lipopolysaccharide of the fish pathogen Aeromonas bestiarum strain P1S

The O-specific polysaccharide obtained by mild-acid degradation of lipopolysaccharide of Aeromonas bestiarum P1S was studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy. The sequence of the sugar residues was determined using ¹H,¹H NOESY and ¹H,¹³C HMBC experiments. The O-specific polysaccharide was found to be a high-molecular-mass polysaccharide composed of tetrasaccharide repeating units of the structureSince small amounts of a terminal Quip3N residue were identified in methylation analysis, it was assumed that the elucidated structure also represented the biological repeating unit of the O-specific polysaccharide.     

Partial purification of glycosyltransferases participating in the biosynthesis of Salmonella anatum and S. kentucky O-antigens using high performance gel chromatography]

The solubilized glycosyltransferases which catalyse the biosynthesis of Salmonella anatum and S. kentucky O-specific polysaccharides were partially purified by HPLC on Superose 12. Two mannosyl transferases from S. kentucky were separated by gel chromatography; these transferases were found useful for chemical-enzymic synthesis of polyprenylpyrophosphate derivatives of trisaccharides Tal-Man-Gal and Man-Tal-Gal.     

Synthesis of the repeating unit of the O-specific polysaccharide of Shigella sonnei and quantitation of its serologic activity

The chemical synthesis of the zwitterionic disaccharide 2 is described that corresponds to the repeating unit of the O-specific polysaccharide (1) of the gram-negative human pathogen Shigella sonnei. Passive hemolysis inhibition tests using a hyperimmune rabbit serum raised against S. sonnei showed that the serologic activity of the disaccharide 2 is nearly 2- to 3-fold higher than those of its component monosaccharides. NMR data of 2 are in support of the proposed structure of the O-specific polysaccharide.     

A new ethanolamine phosphate-containing variant of the O-antigen of Shigella flexneri type 4a

The O-specific polysaccharide (O-antigen) structure of a Shigella flexneri type 4a strain from the Dysentery Reference Laboratory (London, UK) was elucidated in 1978 and its characteristic feature was found to be α-d-glucosylation of GlcNAc at position 6, which defines O-factor IV. Our NMR spectroscopic studies of the O-specific polysaccharides of two other strains belonging to S. flexneri type 4a (G1668 from Adelaide, Australia, and 1359 from Moscow, Russia) confirmed the carbohydrate backbone structure but revealed in both strains an additional component, ethanolamine phosphate (EtnP), attached at position 3 of one of the rhamnose residues:

Full-size image (5K)

Phosphorylation has not been hitherto reported in any S. flexneri O-antigen. Reinvestigation of the O-specific polysaccharide of S. flexneri type 4b showed that it is not phosphorylated and confirmed its structure established earlier.     

Localization of enterobacterial common antigen in Yersinia enterocolitica by the immunoferritin technique.

Rabbits were immunized with the enterobacterial common antigen (ECA)-immunogenic strain Escherichia coli F470. ECA-specific antiserum was obtained by absorbing the resulting antisera with the genetically closely related ECA-negative strain E. coli F1283. These two strains also served as positive and negative controls in the localization study of ECA in Yersinia enterocolitica strain 75, smooth and rough forms (Ye75S and Ye75R), by the indirect immunoferritin technique. Cells of Ye75S grown at 22 degrees C showed no labeling with ferritin after treatment with the ECA-specific antiserum and subsequent ferritin-conjugated goat anti-rabbit antibodies. If the cells were grown at 40 degrees C, however, most of the cells showed weak ferritin labeling. At this higher growth temperature, the lipopolysaccharide of this strain contains less O-specific chains (6-deoxy-L-altrose), as was shown in a previous study. The rough mutant Ye75R, which lacks O-specific chains completely, showed denser labeling with ferritin. These results indicate that ECA on the cell surface of Ye75S is covered by O-specific chains of the lipopolysaccharide if grown at 22 degrees C and is therefore not accessible to ECA antibodies. It becomes accessible, however, when O-chains are lacking (R mutants) or when they are reduced in size or amount (growth at 40 degrees C).     

The distinctive features of the structure of the Pseudomonas fluorescens IMV 247 (biovar II) lipopolysaccharide

The results of the study of the Pseudomonas fluorescens IMV 247 (biovar II) lipopolysaccharide (LPS) isolated from the dry bacterial mass by Westphal's method and purified by repeated ultracentrifugation are presented. The macromolecular organization of the LPS is characterized by the presence of S and R forms of LPS molecules in a 1:1 ratio. The structural components of the LPS molecule--lipid A, the core oligosaccharide, and the O-specific polysaccharide--were isolated and characterized. 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, and dodecanoic acids proved to be the main lipid A fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic portion. Glucose, galactose, arabinose, rhamnose, glucosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulonate (KDO) were revealed in the heterogeneous fraction of the core oligosaccharide. The O-specific polysaccharide chain was composed of repeating tetrasaccharide units consisting of L-rhamnose (L-Rha), 3,6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido-2,4,6-trideoxy-4[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb), and 2-acetamido-2-deoxy-D-galacturonic acid (D-GalNAcA) residues. A peculiarity of the O-specific polysaccharide was that it released, upon partial acid hydrolysis, the nonreducing disaccharide GalNAcA-->QuiNAc4NHb with a 3-hydroxybutyryl group glycosylated intramolecularly with a QuiN4N residue. Double immunodiffusion in agar and lipopolysaccharide precipitation reactions revealed no serological interrelationship between the strain studied and the P. fluorescens strains studied earlier.     

Change in the protective activity of the ribosomal fractions of Salmonella typhimurium in mutations in the rfa region and the transduction replacement of the rfb region]

It was shown that mutation in the rfa region causing disturbances in the structure of the basal part of the polysaccharide of the cell wall or the absence of O-specific side chains led to the loss of protective activity of the ribosomal fractions isolated from the cells of the murine typhoid salmonella by sedimentation with dihydrostreptomycine sulphate. Ribosomal fractions isolated from the murine typhoid salmonella transductants with the replaced rfb region failed to protect the animals from the infection with the virulent. S. typhimurium, S. enteritidis cultures. The virulence of the mutants and transductants was also changed in comparison with the initial strains.     

The molecular heterogeneity of Shigella sonnei lipopolysaccharide based on polyacrylamide gel electrophoretic data

The molecular heterogeneity of S. sonnei lipopolysaccharide (LPS), reflecting the size of lateral O-specific polysaccharide chains, has been established by the method of electrophoresis in acrylamide gel in the presence of sodium dodecyl sulfate and urea. The dominating components fall into three types, viz. those with 0-3, 10-16 and 35-40 repeating structures, the remaining components being minor ones. The electrophoretic profile of S. sonnei LPS considerably differs from the profiles of Escherichia coli and S. flexneri LPS, but coincides with the LPS profiles of other strains with different virulence. The preparations of LPS obtained by extraction with trichloroacetic acid have the same electrophoretic profiles as LPS obtained by the method of aqueous phenol extraction. The domination of certain molecular variants reflects, seemingly, specific features of the biosynthesis of LPS, characteristic of a given strain. The mechanisms of the preferable synthesis of lateral O-specific chains of the definite size and the importance of the molecular parameters of lateral chains for the biological properties of LPS require further study.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.