A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine.

Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast to the previously characterized forms of C-polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This structure is identical to the double choline-substituted form of C-polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C-polysaccharide variants to four.     

The teichoic acid (C-polysaccharide) synthesized by Streptococcus pneumoniae serotype 5 has a specific structure

The teichoic acid synthesized by Streptococcus pneumoniae serotype 5, also known as pneumococcal common antigen (C-polysaccharide), was purified. On the basis of compositional analysis, HPAEC-PAD analysis, MALDI-TOF mass spectrometry and NMR spectroscopy, made on the native polysaccharide and on the dephosphorylated repeating unit, the following structure is proposed: [structure: see text]. This C-polysaccharide (C-PS), differs from those previously described by the replacement of Glc by Gal in its repeating unit structure.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study

Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. These streptococci all coaggregated strongly with both A. viscosus and A. naesludii strains, whereas S. oralis C104 interacted preferentially with certain strains of the latter species. Receptor polysaccharide was isolated from S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The 1H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by 1H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments (1H and 13C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages with the following structure. [----6)Galf(beta 1----3)Galp(beta 1----6)Galf(beta 1----6)GalpNAc(beta 1----3) Galp(alpha 1----1)ribitol(5----PO4-]n     

The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine.

The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.     

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.     

Structure of a new ribitol teichoic acid-like O-polysaccharide of a serologically separate Proteus vulgaris strain, TG 276-1, classified into a new Proteus serogroup O53

An unusual ribitol teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide from a previously non-classified Proteus vulgaris strain TG 276-1. Structural studies using chemical analyses and 2D (1)H and (13)C NMR spectroscopy showed that the polysaccharide is a zwitterionic polymer with a repeating unit containing 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N) and two D-ribitol phosphate (D-Rib-ol-5-P) residues and having the following structure:[formula: see text] where the non-glycosylated ribitol residue is randomly mono-O-acetylated. Based on the unique O-polysaccharide structure and the finding that the strain studied is serologically separate among Proteus bacteria, we propose to classify P. vulgaris strain TG 276-1 into a new Proteus serogroup, O53.     

The structure of a repetitive unit of the glycerolphosphate- containing O-specific polysaccharide chain from Yersinia kristensenii strain 103 (0:12,26) lipopolysaccharide]

Mild acid hydrolysis of the lipopolysaccharide from Yersinia kristensenii strain 103 (0:12.26) afforded teichoic acid-like polysaccharide. From the results of methylation, dephosphorylation, partial Smyth degradation, and 13C and 31P NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [formula: see text] The structure was confirmed by complete interpretation of polysaccharide 13C NMR spectrum.     

Structural determination and immunochemical characterization of the type V group B Streptococcus capsular polysaccharide

The type V capsular polysaccharide of group B Streptococcus has been isolated and purified, and its repeating unit structure determined. The native type V polysaccharide contains D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and sialic acid in a molar ratio of 3:2:1:1. Methylation analysis and 1H NMR and 13C NMR analysis of the native type V polysaccharide and of its specifically degraded products permitted the determination of the repeating unit structure of the type V polysaccharide: [formula: see text] The type V polysaccharide has certain structural features in common with other group B streptococcal capsular polysaccharides but is antigenically distinct: no immunologic cross-reactivity was observed between type V and types Ia, Ib, II, III, or IV polysaccharides. Studies of antibody binding to the partially degraded forms of the type V polysaccharide indicated that the native epitope is complex, involving most if not all of the sugar residues of the repeating unit.     

Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.     

NMR-based identification of cell wall galactomannan of Streptomyces sp. VKM Ac-2125

The major cell wall polymer of Streptomyces sp. VKM Ac-2125, the causative agent of potato scab, is galactomannan with the repeating unit of the following structure: [carbohydrate structure in text] The polysaccharide with such a structure is found in the bacterial cell wall for the first time. The cell wall also contains small amount of a teichoic acid of the poly(glycerol phosphate) type and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid.     

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 a teichoic acid-like O-polysaccharide of Escherichia coli O29

A teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O29. The O-polysaccharide and an oligosaccharide obtained by dephosphorylation of the O-polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The following structure of the branched oligosaccharide repeating unit, containing five monosaccharide residues and glycerol 1-phosphate (D-Gro-1-P), was established: [carbohydrate structure: see text].     

Anionic polymers of the cell wall of Streptomyces sp. VKM An-2534

The cell wall of Streptomyces sp. VKM An-2534, the causative agent of common scab in potato tubers, which does not synthesize thaxtomin and is phylogenetically close to phytopathogen Streptomyces setonii sp. ATCC 25497, contains two anionic carbohydrate-containing polymers. The major polymer is teichuronic acid, whose repeating unit is disaccharide --> 4)-beta-D-ManpNAc3NAcyA-(1 --> 3)-alpha-D-GalpNAc-(1-->, where Acy is a residue of acetic or L-glutamic acid. The polymer of such structure has been found in Gram-positive bacteria for the first time. The minor polymer is teichoic acid [1,5-poly(ribitol phosphate)], in which a part of the ribitol residues are glycosylated at C4 with beta-D-Glcp and, probably, with beta-D-GlcpNAc and some residues are O-acylated with Lys residues. The structures were proved by chemical and NMR spectroscopic methods. It is likely that the presence of acidic polysaccharides on the surface of the phytopathogenic streptomycete is necessary for its attachment to the host plant.     

Structure of the O-polysaccharide of Providencia alcalifaciens O8 containing (2S,4R)-2,4-dihydroxypentanoic acid, a new non-sugar component of bacterial glycans

A glycerol teichoic acid-like O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O8 and studied by chemical methods and NMR spectroscopy, including 2D ROESY, {(1)H,(13)C} HSQC, and HMQC-TOCSY experiments. It was found that the compound contains a new component of bacterial lipopolysaccharides: ether-linked (2S,4R)-2,4-dihydroxypentanoic acid (Dhpa), which was identified by NMR spectroscopy. The following structure of the repeating unit of the polysaccharide was established:     

Application of high-resolution n.m.r. spectroscopy to the elucidation of the structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 7F

The specific capsular polysaccharide of Streptococcus pneumoniae type 7F (American type 51) is a high-molecular-weight neutral polymer composed of 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, L-rhamnose, and 2-O-acetyl-L-rhamnose residues. N.m.r. spectroscopy (1H and 13C), in conjunction with composition and methylation analyses, and periodate oxidation data, showed the polysaccharide to be a branched polymer with a repeating heptasaccharide unit having the following structure. (formula; see text)     

Carbohydrate-containing polymers of the cell wall of the thermophilic streptomycete Streptomyces thermoviolaceus subsp. thermoviolaceus VKM Ac-1857T

Anionic polymers of the cell surface of a thermophilic streptomycete were investigated. The cell wall of Streptomyces thermoviolaceus subsp. thermoviolaceus VKM Ac-1857(T) was found to contain polymers with different structure: teichoic acid--1,3-poly(glycerol phosphate), disaccharide-1-phosphate polymer with repeating unit -6)-alpha-Galp-(1-->6)-alpha-GlcpNAc-P-, and polysaccharide without phosphate with repeating unit -->6)-alpha-GalpNAc-(1-->3)-beta-GalpNAc-(1-->. Disaccharide-1-phosphate and polysaccharide without phosphate have not been described earlier in prokaryotic cell walls.     

Versatility of choline metabolism and choline-binding proteins in Streptococcus pneumoniae and commensal streptococci

The pneumococcal choline-containing teichoic acids are targeted by choline-binding proteins (CBPs), major surface components implicated in the interaction with host cells and bacterial cell physiology. CBPs also occur in closely related commensal species, Streptococcus oralis and Streptococcus mitis , and many strains of these species contain choline in their cell wall. Physiologically relevant CBPs including cell wall lytic enzymes are highly conserved between Streptococcus pneumoniae and S. mitis . In contrast, the virulence-associated CBPs, CbpA, PspA and PcpA, are S. pneumoniae specific and are thus relevant for the characteristic properties of this species.     

A novel teichoic acid from the cell wall of Streptomyces sp. VKM Ac-2275

The cell wall of a pathogenic strain Streptomyces sp. VKM Ac-2275 isolated from potato tubers infected by scab contains a teichoic acid related to poly(glycosylpolyol phosphate) with a repeating unit established by chemical and NMR spectroscopic methods. About 60% of l-rhamnose residues bear an O-acetyl group at O-2 and 20% of the internal glucose residues contain an additional phosphate at C-4. The polymer is built of 5-6 units. This structure is found in bacteria for the first time. The strain is phylogenetically closest to the scab-causing species Streptomyces scabiei and Streptomyces europaeiscabiei, but differs from both these species in morphological and physiological characters and does not produce thaxtomin A, the main phytotoxin produced by S. scabiei.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: a receptor for lectin-mediated interbacterial adherence

Lectin-carbohydrate binding is known to play an important role in a number of different cell-cell interactions including those between certain species of oral streptococci and actinomyces that colonize teeth. The cell wall polysaccharides of Streptococcus oralis ATCC 10557, S. oralis 34, and Streptococcus mitis J22, although not identical antigenically, each function as a receptor molecule for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii. Carbohydrate analysis of the receptor polysaccharide isolated from S. oralis ATCC 10557 shows galactose (3 mol), glucose (1 mol), GalNAc (1 mol), and rhamnose (1 mol). 1H NMR spectra of the polysaccharide show that is is partially O-acetylated. Analysis of the 1H NMR spectrum of the de-O-acetylated polysaccharide shows that it is composed of repeating subunits containing six monosaccharides and that the subunits are joined by a phosphodiester linkage. The 1H and 13C NMR spectra were completely assigned by two-dimensional homonuclear correlation methods and by 1H-detected heteronuclear multiple-quantum correlation (1H[13C]HMQC). The linkage of the component monosaccharides in the polymer, deduced from two-dimensional 1H-detected heteronuclear multiple-bond correlation spectra (1H[13C]HMBC), shows that the repeating unit of the de-O-acetylated polymer is a linear hexasaccharide with no branch points. The complete 1H and 13C assignment of the native polysaccharide was carried out by the same techniques augmented by a 13C-coupled hybrid HMQC-COSY method, which is shown to be especially useful for carbohydrates in which strong coupling and overlapping peaks in the 1H spectrum pose difficulties. The fully assigned spectra of the native polymer show that each of two different positions is acetylated in one-third of the repeating subunits and that the acetylation is randomly distributed along the polymer. The exact positions of acetylation were assigned by a carbonyl-selective HMBC method that unambiguously defines the positions of O-acetylation. The complete structure of the native polysaccharide in S. oralis ATCC 10557 is [formula: see text] Comparison of this structure with those previously determined for the polysaccharides of strains 34 and J22 suggests that the similar lectin receptor activities of these molecules may depend on internal galactofuranose linked (beta 1----6)- to Gal(beta 1----3)GalNAc(alpha) or GalNAc(beta 1----3)Gal(alpha).     

Molecular and antigenic characterization of a Streptococcus oralis coaggregation receptor polysaccharide by carbohydrate engineering in Streptococcus gordonii

The coaggregation receptor polysaccharides (RPS) of Streptococcus oralis and related species are recognized by lectin-like adhesins on other members of the oral biofilm community and by RPS-specific antibodies. The former interactions involve beta-GalNAc or beta-Gal containing host-like motifs in the oligosaccharide repeating units of these polysaccharides, whereas the latter involves features of these molecules that are immunogenic. In the present investigation, the molecular and corresponding structural basis for the serotype specificity of S. oralis ATCC 10557 RPS was determined by engineering the production of this polysaccharide in transformable Streptococcus gordonii 38. This involved the systematic replacement of genes in the rps cluster of strain 38 with different but related genes from S. oralis 10557 and structural characterization of the resulting polysaccharides. The results identify four unique genes in the rps cluster of strain 10557. These include wefI for an alpha-Gal transferase, wefJ for a GalNAc-1-phosphotransferase that has a unique acceptor specificity, wefK for an acetyl transferase that acts at two positions in the hexasaccharide repeating unit, and a novel wzy associated with the beta1-3 linkage between these units. The serotype specificity of engineered polysaccharides correlated with the wefI-dependent presence of alpha-Gal in these molecules rather than with partial O-acetylation or with the linkage between repeating units. The findings illustrate a direct approach for defining the molecular basis of polysaccharide structure and antigenicity.