Structural studies of the O-antigenic polysaccharide from Escherichia coli O177.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with (1)H and (13)C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by (1)H,(13)C-heteronuclear multiple-bond correlation and (1)H,(1)H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure: →2)-α-L-Rhap-(1→3)-α-L-FucpNAc-(1→3)-α-L-FucpNAc-(1→3)-ß-D-GlcpNAc-(1→. An α-L-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the (1)H and (13)C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-D-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-L-FucpNAc-(1→3)-ß-D-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.

Complete (1)H and (13)C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper.

The computer program casper uses (1)H and (13)C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the (1)H and (13)C, as well as (31)P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and ß-anomeric forms. In total 35 (1)H and (13)C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D (1)H,(13)C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t(1) incremented (1)H,(13)C-HSQC experiment and a 1D (1)H,(1)H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3Hz apart. The (1)H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental (1)H and (13)C NMR chemical shifts.

Structural studies of the capsular polysaccharide produced by Leuconostoc mesenteroides ssp. cremoris PIA2.

The structure of the capsular polysaccharide (CPS) produced by Leuconostoc mesenteroides ssp. cremoris PIA2 has been determined using component analysis and NMR spectroscopy. (1)H and (13)C resonances were assigned using 2D NMR experiments, and sequential information was obtained by (1)H,(1)H-NOESY and (1)H,(13)C-HMBC experiments. The CPS consists of linear pentasaccharide repeating units with the following structure: →3)-ß-D-Galf-(1→6)-ß-D-Galf-(1→2)-ß-D-Galf-(1→6)-ß-D-Galf-(1→3)-ß-D-Galp-(1→, in which four out of the five sugar residues have the furanoid ring form, a structural entity found in bacteria but not in mammals. The analysis of the magnitude of the homonuclear three-bond coupling constants of the anomeric protons for the five-membered sugar rings indicates that the sugar residues substituted at a primary carbon atom show one kind of conformational preferences, whereas those substituted at a secondary carbon atom show another kind of conformational preferences.

Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O175.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O175 has been elucidated. Component analysis together with (1)H and (13)C NMR spectroscopy experiments were used to determine the structure. Inter-residue correlations were determined by (1)H,(1)H-NOESY, and (1)H,(13)C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: →2)-α-D-Glcp-(1→4)-α-D-GlcpA-(1→3)-α-D-Manp-(1→2)-α-D-Manp-(1→3)-ß-D-GalpNAc-(1→ Cross-peaks of low intensity from an α-linked glucopyranosyl residue were present in the (1)H,(1)H-TOCSY NMR spectra. The α-D-Glcp residue is suggested to originate from the terminal part of the polysaccharide and consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end. The repeating unit of the E. coli O175 O-antigen is similar to those from E. coli O22 and O83, both of which carry an α-D-Glcp-(1→4)-D-GlcpA structural element, thereby explaining the reported cross-reactivities between the strains.

Brucella melitensis 16M produces a mannan and other extracellular matrix components typical of a biofilm.

Mutations in the Brucella melitensis quorum-sensing (QS) system are involved in the formation of clumps containing an exopolysaccharide. Here, we show that the overexpression of a gene called aiiD in B. melitensis gives rise to a similar clumping phenotype. The AiiD enzyme degrades AHL molecules and leads therefore to a QS-deficient strain. We demonstrated the presence of exopolysaccharide and DNA, two classical components of extracellular matrices, in clumps produced by this strain. We also observed that the production of outer membrane vesicles is strongly increased in the aiiD-overexpressing strain. Moreover, this strain allowed us to purify the exopolysaccharide and to obtain its composition and the first structural information on the complex exopolysaccharide produced by B. melitensis 16M, which was found to have a molecular weight of about 16 kDa and to be composed of glucosamine, glucose and mostly mannose. In addition, we found the presence of 2- and/or 6-substituted mannosyl residues, which provide the first insights into the linkages involved in this polymer. We used a classical biofilm attachment assay and an HeLa cell infection model to demonstrate that the clumping strain is more adherent to polystyrene plates and to HeLa cell surfaces than the wild-type one. Taken together, these data reinforce the evidence that B. melitensis could form biofilms in its lifecycle.

Genetic and structural relationships of Salmonella O55 and Escherichia coli O103 O-antigens and identification of a 3-hydroxybutanoyltransferase gene involved in the synthesis of a Fuc3N derivative.

O-antigen (O-polysaccharide), a part of the outer membrane of Gram-negative bacteria, is one of the most variable cell constituents and is related to bacterial virulence. O-antigen diversity is almost entirely due to genetic variations in O-antigen gene clusters. In this study, the O-polysaccharide structures of Salmonella O55 and Escherichia coli O103 were elucidated by chemical analysis and nuclear magnetic resonance spectroscopy. It was found that the O-polysaccharides have similar pentasaccharide O-units, which differ only in one sugar (glucose versus N-acetylglucosamine) and in the N-acyl group (acetyl versus 3-hydroxybutanoyl) on 3-amino-3,6-dideoxy-d-galactose (d-Fuc3N). The Salmonella O55 antigen gene cluster was sequenced and compared with the E. coli O103 antigen gene cluster reported previously. The two gene clusters were found to share high-level similarity (DNA identity ranges from 53% to 76%), except for two putative acyl transferase genes (fdtC in Salmonella O55 and fdhC in E. coli O103) which show no similarity. Replacement of the fdtC gene in Salmonella O55 with the fdhC gene from E. coli O103 resulted in production of a modified O-antigen, which contains a 3-hydroxybutanoyl derivative of Fuc3N in place of 3-acetamido-3,6-dideoxygalactose. This finding strongly suggests that fdhC is a 3-hydroxybutanoyltransferase gene. The sequence similarity level suggested that the O-antigen gene clusters of Salmonella O55 and E. coli O103 originate from a common ancestor, and this evolutionary relationship is discussed.