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.     

The chemical structure of a fragment of Micrococcus lysodeikticus cell-wall.

A fragment of Micrococcus lysodeikticus cell-wall obtained by cetylpyridinium recipitation from the nondialyzable portion of the degradation products of egg-white lysozyme was studied by the periodate oxidation and methylation procedures. The fragment consists of a polysaccharide chain composed of about 40 repeating (1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranosyluronic acid)-(1 leads to 6)-O-(alpha-D-glucopyranosyl) residues with D-glucopyranosyl residues at both ends. The alpha-D-glucopyranose residue at the reducing end is linked to a phosphate group that is also linked to C-6 of a 2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl residue of a peptidoglycan chain composed of four repeating (1 leads to 4)-O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl] residues. The peptidoglycan chain has, as nonreducing group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, and, as reducing residue, a 2-acetamido-3-O-(D-1-carboxytheyl)-2-deoxy-beta-D-glucose residue.     

The teichuronic acid from the walls of Bacillus licheniformis A.T.C.C. 9945.

The teichuronic acid of Bacillus licheniformis A.T.C.C. 9945 grown under phosphate limitation was isolated from the cell walls and purified by ion-exchange and Sephadex chromatography. The detailed structure of the polysaccharide was established by methylation analysis, periodate oxidation and partial acid hydrolysis. The polymer is composed of tetrasaccharide repeating units with the structure [GlcA beta(1 leads to 4)GlcA beta(1 leads to 3)GalNAc beta(1 leads to 6)GalNAc alpha(1 leads to 4)n. 13C n.m.r. analysis has confirmed most of the structural features of the polysaccharide and, in particular, the anomeric configurations and linkage positions of substituents. The teichuronic acid from glucose-limited cells was identical with that from cells grown under phosphate limitation.     

Primary structure of the Escherichia coli serotype K42 capsular polysaccharide and its serological identity with the Klebsiella K63 polysaccharide.

The Escherichia coli K42 capsular polysaccharide consists of leads to 3)-alpha-D-Galp-(1 leads to 3)-alpha-D-GalUAp-(1 leads to 3)-alpha-L-Fucp-(1 leads to repeating units. The E. coli K42 and Klebsiella K63 antigens are serologically identical.     

Structural analysis and chemical depolymerization of the capsular polysaccharide of Streptococcus pneumoniae type 1

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T(2) relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages.     

Somatic antigens of Shigella and Escherichia coli. Determination of the structure of O-specific polysaccharide from Shigella boydii type 12 and its serological properties compared with the O-specific polysaccharide from Escherichia coli

The structure of the O-specific polysaccharide of the somatic antigen (lipopolysaccharide) of Shigella boydii, type 12, was established by 1H- and 13C-NMR, methylation analysis and partial acid hydrolysis methods. The polysaccharide consists of pentasaccharide repeating units of the following structure: (formula; see text) The amount of O-acetyl groups was far less than stoichiometric, only about 2 for 3-4 repeating units. Nevertheless, the results of serological studies revealed 3-O-acetyl-alpha-L-rhamnose residue to be the major immunodominant group. In spite of the presence of similar trisaccharide fragments, the lipopolysaccharide and polysaccharide from Shigella boydii type 12 gave no crossreaction with lipopolysaccharide and polysaccharide from Escherichia coli 07. The possible reasons of the absence of serological relatedness between the Sh. boydii, type 12, and E. coli 07 cells were discussed.     

Structure of the K95 antigen from Escherichia coli O75:K95:H5, a capsular polysaccharide containing furanosidic KDO-residues

The structure of the K95 antigenic capsular polysaccharide (K95 antigen) of Escherichia coli O75:K95:H5 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and methylation analysis. The K95 polysaccharide, which contains furanosidic 3-deoxy-D-manno-2-octulosonic acid (KDOf) residues, consists of----3)-beta-D-Rib-(1----8)-KDOf-(2----repeating units, has a molecular weight of approximately 25,000 (approximately 65 repeating units), and is randomly O-acetylated (1 acetyl group per repeating unit at unknown positions).     

Structure of the serotype f polysaccharide antigen of Streptococcus mutans

The structure of the serotype f polysaccharide antigen of Streptococcus mutans was determined by methylation analysis, periodate oxidation, and partial methanolysis, and the configuration of the anomeric linkages by 13C-n.m.r. spectroscopy, indicating the trisaccharide repeating unit----3)-alpha-L-Rhap-(1----2)-[alpha-D-Glcp-(1----3)]-alpha-L-+ ++Rhap- (1----. The structure of the backbone of the polysaccharide was confirmed by demonstrating immunological identity between the product of Smith degradation of the S. mutans serotype f antigen and the group A-variant streptococcal polysaccharide.     

Structural determination of the capsular polysaccharide of Neisseria meningitidis group I: a two-dimensional NMR analysis

The capsular polysaccharide antigen of Neisseria meningitidis group I was isolated by Cetavlon precipitation and purified by ion-exchange chromatography. The structure of the I polysaccharide was determined largely by comprehensive proton and carbon-13 nuclear magnetic resonance studies in which both one-dimensional and two-dimensional experiments were carried out directly on the I polysaccharide. The I polysaccharide is composed of the repeating unit----4)alpha-L-GulpNAcA(1----3)[4-OAc]beta-D-ManpNA-cA(-- --in which the former residue adopts the 4C1 (L) conformation and the latter residue adopts the 4C1 (D) conformation. The one-bond coupling between the anomeric carbon and proton (1J13C,H) of the 2-acetamido-2-deoxy-beta-D-mannuronopyranosyl residue is not consistent with its beta-D configuration. This anomalous value of 1J13C,H for this residue is due to through-space anisotropy effects on its anomeric proton, generated by the proximity of the carboxyl group of the neighboring 2-acetamido-2-deoxy-alpha-L-guluronopyranosyl residue. The O-acetyl substituents of the I polysaccharide are essential for its antigenicity to group I polysaccharide-specific antibodies.     

Klebsiella serotype-13 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase

Periodate oxidation and Smith degradation, methylation analysis including uronic acid degradation, partial hydrolysis with acid, bacteriophage degradation, and p.m.r. spectroscopy have been used to elucidate the primary structure of the Klebsiella serotype-13 capsular polysaccharide. The polymer consists of pentasaccharide repeating-units comprising a 4)-beta-D-Manp-(1 leads to 4)-alpha-D-Glcp-(1 leads to 3)-beta-D-Glcp-(1 leads to chain with a 3,4-O-(1-carboxyethylidene)-beta-D-Galp-(1 leads to 4)-alpha-D-GlcAp-(1 leads to branch at position 3 of the mannose. It is shown that there is a glycanase activity associated with particles of Klebsiella bacteriophage No. 13, which catalyses hydrolysis of chain beta-D-Glcp-(1 leads to 4)-beta-D-Manp linkages in the type-13 polysaccharide. The chemical basis of some serological cross-reactions of the Klebsiella K13 antigen is discussed.     

A new model of pneumococcal lipoteichoic acid structure resolves biochemical, biosynthetic, and serologic inconsistencies of the current model

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(beta1-->3)AATGal(beta1-->3)Glc(alpha1-->3)-acyl(2)Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(alpha1-->3)GalNAc(beta1-->, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(alpha1-->3)-acyl(2)Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.     

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-->     

Structure of the 2-keto-3-deoxy-D-manno-octonic-acid-containing capsular polysaccharide (K12 antigen) of the urinary-tract-infective Escherichia coli O4:K12:H-

The primary structure of the K12 antigenic capsular polysaccharide (K12 antigen) of Escherichia coli O4:K12:H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation and oligosaccharide analysis. The polysaccharide consists of repeating trisaccharide alpha-rhamnosyl-1,2-alpha-rhamnosyl-1,5-dOclA units (dOclA = 2-keto-3-deoxy-D-manno-octonic acid) which are joined through beta-2,3-linkages. About 50% of the dOclA units are O-acetylated at C7 or C8. The sequence of acetylated and non-acetylated dOclA residues is not known. As had been reported before, the polysaccharide is linked to a phosphatidic acid at the reducing end (dOclA) via a phosphodiester bridge. The serologically specific part of the K12 antigen is its polysaccharide moiety.     

Structure of the fructose-containing K52 capsular polysaccharide of uropathogenic Escherichia coli O4:K52:H-

The chemical structure of the K52 antigenic capsular polysaccharide (K52 antigen) of Escherichia coli O4:K52:H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation before and after graded acid hydrolysis and by oligosaccharide analysis. The polysaccharide consists of a backbone of alpha-galactose units interlinked between C1 and C3 by phosphodiester bridges. This poly(alpha-galactosyl-phosphate) is substituted at C2 of each galactose unit by beta-fructofuranose residues. About 80% of the galactose units are O-acetylated at C4 and about 10% of the fructose units are both O-acetylated and O-propionylated at C1. The K52 polysaccharide has an average molecular mass of 34 kDa, thus consisting of approximately 65 fructosyl-galactosyl-phosphate repeating units.     

Structural elucidation of the Brucella melitensis M antigen by high-resolution NMR at 500 MHz

The Brucella M antigen from the species type strain Brucella melitensis 16M has been identified as a component of the cell wall lipopolysaccharide (LPS). O polysaccharide liberated from this LPS by mild acid hydrolysis exhibited M activity in serological tests and was shown to be a homopolymer of 4-formamido-4,6-dideoxy-alpha-D-mannopyranosyl residues arranged in an oligosaccharide repeating unit as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native lipopolysaccharide. Structural analysis of the O polysaccharide by NMR methods was difficult due to apparent microheterogeneity of the repeating unit, which was in fact caused by the presence of rotational isomers of the N-formyl moiety. This problem was resolved by chemical modification of the polysaccharide to its amino and N-acetyl derivatives, the 500-MHz 1H and 125-MHz 13C NMR spectra of which could be analyzed in terms of a unique structure through application of pH-dependent beta-shifts and two-dimensional techniques that included COSY, relayed COSY, and NOESY experiments together with heteronuclear C/H shift correlation spectroscopy. On the basis of these experiments and supported by methylation and periodate oxidation data, the structure of the M polysaccharide was determined as a linear polymer of unbranched pentasaccharide repeating units consisting of four 1,2-linked and one 1,3-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues. The marked structural similarity of the M antigen and the A antigen, which is known to be a 1,2-linked homopolysaccharide of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units, accounts for cross-serological reactions of the two and the long-standing confusion surrounding the nature of their antigenic determinants. Structural and serological considerations in conjuction with the sodium dodecyl sulfate banding pattern of Brucella A LPS suggest that its biosynthesis differs appreciably from that of the M antigen, which appears to be synthesized by regulated assembly of preformed oligosaccharide repeating units. Temperature, lysogenic phage may be responsible for such biosynthetic and structural variations.     

Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).

The lipopolysaccharide (LPS) of Klebsiella serotype O2 is antigenically heterogeneous; some strains express multiple antigenic factors. To study this heterogeneity, we determined the structure of the O-antigen polysaccharides in isolates belonging to serotypes O2(2a), O2(2a,2b), and O2(2a,2c), by using composition analysis, methylation analysis, and both 1H and 13C nuclear magnetic resonance spectroscopy. The repeating unit structure of the 2a polysaccharide was identified as the disaccharide [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] and was identical to D-galactan I, one of two O polysaccharides present in the LPS of Klebsiella pneumoniae serotype O1 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). LPS from serotype O2(2a,2b) also contained D-galactan I as the only O polysaccharide, suggesting that the 2b antigen is not an O antigen. The LPS of serotype O2(2a,2c) contained a mixture of two structurally distinct O polysaccharides and provides a second example of this phenomenon in Klebsiella spp. One polymer was identical to D-galactan I, and the other polysaccharide, the 2c antigen, was a polymer with a disaccharide repeating unit structure, [----3)-beta-D-GlcpNAc-(1----5)-beta-D-Galf-(1----]. The 2c structure does not resemble previously reported O polysaccharides from Klebsiella spp. Periodate oxidation confirmed that D-galactan I and the 2c polysaccharide are distinct glycans, rather than representing domains within a single polysaccharide chain. Monoclonal antibodies against the 2c antigen indicated that only LPS molecules with the longest O-polysaccharide chains contained the 2c epitope.     

Structural analysis of the surface polysaccharide of Staphylococcus aureus M.

The chemical structure of the surface polysaccharide from Staphylococcus aureus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl-(1 leads to 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-D-aminogalacturonic acid via an amide bond.     

Structural characterization and MHCII-dependent immunological properties of the zwitterionic O-chain antigen of Morganella morganii

Morganella morganii is a commensal Gram-negative bacterium that has long been known to produce an antigen bearing phosphocholine groups. We determined the structure of this O-chain antigen and found that its repeating unit also contains a free amino group and a second phosphate: This alternating charge character places the M. morganii O-chain polysaccharide into a small family of zwitterionic polysaccharides (ZPSs) known to induce T-cell-dependent immune responses via presentation by class II major histocompatibility complex (MHCII) molecules. In vitro binding assays demonstrate that this O-chain interacts with MHCII in a manner that competes with binding of the prototypical ZPS antigen PSA from Bacteroides fragilis, despite its lack of a helical structure. Cellular studies also showed that the M. morganii polysaccharide induces activation of CD4(+) T-cells. Antibody binding experiments using acid hydrolyzed fragments representing the monomer and higher oligomers of the repeating unit showed that the phosphocholine group was the dominant element of the epitope with an overall affinity (K(D)) of about 5 × 10(-5) M, a typical value for an IgM anti-carbohydrate antibody but much lower than the affinity for phosphocholine itself. These data show that the structure of the M. morganii polysaccharide contains a unique zwitterionic repeating unit which allows for immune recognition by T-cells, making it the first identified T-cell-dependent O-chain antigen.     

Cell-wall lipopolysaccharide of the 'Shigella-like' Escherichia coli 058. Structure of the polysaccharide chain.

Two lipopolysaccharide preparations were obtained from Escherichia coli 058 by extraction with 45% aqueous phenol and fractional precipitation with cetyltrimethyl ammonium bromide (Cetavlon). Chemical analysis and polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. The O-specific polysaccharide was characterized with proton magnetic resonance and infrared spectroscopy, optical rotation and paper electrophoresis. Using gas-liquid chromatography and ion-exchange chromatography, it was shown to contain D-mannose, 2-acetamido-2-deoxy-D-glucose, 3-O-(R-1'-carboxyethyl)-L-rhamnose (rhamnolactylic acid), and O-acetyl groups in the molar ratios of 2:1:1:1. The polysaccharide and oligosaccharides obtained from it were subjected to methylation and chromic acid oxidation. The results obtained indicated that the polysaccharide consists of tetrasaccharide repeating units in which the trisaccharide beta-GlcNAc1 - 4alphaMan-1 - 4(2/3-O-Ac)-Man is substituted at C-3 of the non-acetylated mannose with rhamnolactylic acid. The repeating units are joined through alpha-mannosyl-1 - 3-glucosamine bonds. This structure is identical with that of the cell wall polysaccharide of Shigella dysenteriae type 5.     

Synthesis of 6-aminohexylglycosides of a polysaccharide from Streptococcus group A and its fragments

Polycondensation of 4-O-benzoyl-1,2-O-(1-cyanoethylidene)-3-O-(3,4-di-O-benzoyl-2-O-tr ityl-alpha-L- rhamnopyranosyl)-beta-L-rhamnopyranose in the presence of 6-phthalimidohexyl-3,4-di-O-benzoyl-2-O-trityl-alpha-L- rhamnopyranoside affords, after deprotection, the polysaccharide built up of the repeating dissaccharide units----2) Rha (alpha 1----3) Rha (alpha 1----and containing 6-aminohexyl residue at the reducing end. This polysaccharide possesses the structure of the group A-variant streptococcal polysaccharide. Synthesis of 6-aminohexyl glycosides of 2- and 3-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranoses, which corresponds to the repeating units of the above polysaccharide, is described.