Structure determination of Streptococcus suis serotype 9 capsular polysaccharide and assignment of functions of the cps locus genes involved in its biosynthesis.

Streptococcus suis serotype 9 is the most prevalent S. suis serotype in several European countries. In spite of its pathogenicity for pigs and increasing zoonotic potential, limited information is available on this serotype. Here we determined for the first time the chemical composition and structure of serotype 9 capsular polysaccharide (CPS), a major bacterial virulence factor and the antigen at the origin of S. suis classification into serotypes. Chemical and spectroscopic data gave the repeating unit sequence: [3)Glcol-6-P-3-[D-Gal(α1-2)]D-Gal(ß1-3)D-Sug(ß1-3)L-Rha(α1-)]n. Compared to previously characterized S. suis CPSs (serotypes 1, 1/2, 2 and 14), serotype 9 CPS does not contain sialic acid but contains a labile 4-keto sugar (2-acetamido-2,6-dideoxy-ß-D-xylo-hexopyranos-4-ulose), one particular feature of this serotype. A correlation between S. suis serotype 9 CPS sequence and genes of this serotype cps locus encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit was tentatively established. Knowledge of CPS structure and composition will contribute to better dissect the role of this bacterial component in the pathogenesis of S. suis serotype 9.

The structure of the extracellular teichoic acids from the allergy-protective bacterium Lactococcus lactis G121.

The Gram-positive bacterium Lactococcus lactis G121 is a farm isolate that protects mice from ovalbumin-induced asthma. To understand the molecular mechanisms of such allergy-protective properties, the isolation and characterization of cell envelope constituents is crucial. Here, structural analyses of the extracellular teichoic acid (EC TA) from L. lactis G121 are presented. Extraction with 0.9% saline afforded a crude TA fraction. Consecutive size exclusion chromatography on Biogel P60 and P10 matrix was performed to purify the sample. Chemical component analyses, high-resolution electrospray ionization Fourier-transformed ion cyclotron mass spectrometry, and nuclear magnetic resonance spectroscopy were conducted for structural elucidation. The EC TA was a poly(glycosylglycerol phosphate) molecule with a repeating unit of -6)-[ß-D-Glcp-(1→3)-][α-D-GlcpNAc-(1→4)-]α-D-GalpNAc-(1→3)-ß-D-GlcpNAc-(1→2)-glycerol-(1-P-).

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production.

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in non-mucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that 11 psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating pentasaccharide containing d-mannose, d-glucose and l-rhamnose: [See text]. In addition, we identified the sugar nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP-d-mannose, UDP-d-glucose and dTDP-l-rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that wbpW restored Psl production in a pslB mutant and pslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of P. aeruginosa.

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain.

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.

Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]). In the present paper we present an improved procedure for preparation of PIA that conserves its labile substituents and avoids contamination with by-products. Based on structural analysis of the polysaccharide antigens and a thorough overview of the previously published data, we concluded that PIA from S. epidermidis is structurally identical to the recently described poly-beta-(1-->6)-N-acetylglucosamine from PS/A-overproducing strain S. aureus MN8m. We also show that another carbohydrate-containing polymer, extracellular teichoic acid (EC TA), is an essential component of S. epidermidis RP62A biofilms. We demonstrate that the relative amounts of extracellular PIA and EC TA produced depend on the growth conditions. Moderate shaking or static culture in tryptic soy broth favors PIA production, while more EC TA is produced in brain heart infusion medium.

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus epidermidis RP62A, a reference biofilm-positive strain.

The ability to adhere to artificial surfaces and form biofilms is considered as a virulence factor of Staphylococcus epidermidis, one of the major causes of nocosomial infections, especially those related to implanted medical devices. Cell-wall teichoic acid is known to play an important role in biofilm formation of staphylococci. The structure of the cell wall and extracellular teichoic acids of S. epidermidis RP62A, a reference biofilm-positive strain, was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Their structures were found to be a (1-->3)-linked poly(glycerol phosphate), substituted at the 2-position of glycerol residues with alpha-Glc, alpha-GlcNAc, D-Ala and alpha-Glc6Ala. D-Alanyl acylation of a sugar hydroxyl group seems to be a novel structural feature of teichoic acids from staphylococci.