Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 522/C1 and the international type strain from Escherichia coli O 178

The structure of the O-antigenic polysaccharide (PS) from the enteroaggregative Escherichia coli strain 522/C1 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy techniques were used to elucidate 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: [ structure: see text]. Analysis of NMR data reveals that on average the PS consists of four repeating units and indicates that the biological repeating unit contains an N-acetylgalactosamine residue at its reducing end. Serotyping of the E. coli strain 522/C1 showed it to be E. coli O 178:H7. Determination of the structure of the O-antigen PS of the international type strain from E. coli O 178:H7 showed that the two polysaccharides have identical repeating units. In addition, this pentasaccharide repeating unit is identical to that of the capsular polysaccharide from E. coli O9:K 38, which also contains O-acetyl groups.     

Structural and immunochemical relationship between the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 396/C-1 and Escherichia coli O126

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 396/C-1 has been determined. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy were the main methods used. Inter-residue correlations were determined by 1H,1H-NOESY, 1H,13C-heteronuclear multiple-bond correlation and dipole-dipole cross-correlated relaxation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. Analysis of NMR data reveals that on average the PS consists of approximately 13 repeating units and indicates that the biological repeating unit contains an N-acetylglucosamine residue at its reducing end. This structure is different to that reported for the O-antigen polysaccharide from E. coli O126. Monospecific anti-E. coli O126 rabbit serum from The International Escherichia and Klebsiella Centre did not distinguish between the E. coli strain 396/C-1 and the E. coli O126 reference strain, neither in slide agglutination nor in an indirect enzyme immunoassay. Subsequent successful serotyping of the E. coli strain 396/C-1 showed it to be E. coli O126:K+:H27.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.     

Structural elucidation of the O-antigenic polysaccharide from the enteroaggregative Escherichia coli strain 180/C3 and its immunochemical relationship with E. coli O5 and O65

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 180/C3 has been determined. Sugar and methylation analysis together with (1)H and (13)C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure: -->2)beta-D-Quip3NAc-(1-->3)beta-D-RIBf-(1-->4)beta-D-Galp-(1-->3)alpha-D-GalpNAc-(1-->. Analysis of NMR data indicates that the presented sequence of sugar residues also represents the biological repeating unit of the O-chain. The structure is closely related to that of O-antigen polysaccharide from E. coli O5 and partially to that of E. coli O65. The difference between the O-antigen from the 180/C3 strain and that of E. coli O5 is the linkage to the D-Quip3NAc residue, which in the latter strain is 4-O-substituted. The E. coli O65 O-antigen contains as part of its linear pentasaccharide repeating unit a similar structural element, namely -->4)-beta-d-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->2)-beta-D-Quip3NAc-(1-->, thereby indicating that a common epitope could be present for the two polysaccharides. Monospecific anti-E. coli O5 rabbit serum did not distinguish between the two positional isomeric structures neither in slide agglutination nor in an indirect enzyme immunoassay. The anti-O65 serum did react with both the 180/C3 and O5 LPS showing a partial cross-reactivity.     

Structural determination of the O-antigenic polysaccharide from Escherichia coli O166

The O-antigen of the lipopolysaccharide from Escherichia coli O166 has been determined by component analysis together with 1D and 2D NMR spectroscopy techniques. The polysaccharide has pentasaccharide repeating units consisting of D-glucose (1), D-galactose (2) and N-acetyl-D-galactosamine (2) with the following structure: [STRUCTURE: SEE TEXT]. In the 1H NMR, spectrum resonances of low intensity were observed. Further analysis of these showed that they originate from the terminal part of the polysaccharide, thereby revealing that the repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.     

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 ¹H and ¹³C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by ¹H,¹³C-heteronuclear multiple-bond correlation and ¹H,¹H-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)-β-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 ¹H and ¹³C 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)-β-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.     

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 ¹H and ¹³C NMR spectroscopy experiments were used to determine the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY, and ¹H,¹³C-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)-β-d-GalpNAc-(1→Cross-peaks of low intensity from an α-linked glucopyranosyl residue were present in the ¹H,¹H-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.     

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. 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 O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).     

Structural studies of the O-antigenic polysaccharides from Shigella dysenteriae type 3 and Escherichia coli O124, a reinvestigation

The structures of the O-antigenic part of the lipopolysaccharides from Shigella dysenteriae type 3 and Escherichia coli O124 have been reinvestigated. (1)H and (13)C NMR spectroscopy in combination with selected 2D NMR techniques were used to determine the O-antigen pentasaccharide repeating units with the following structure: [see text]. From biosynthetic considerations this should also be the biological repeating unit. The structures of the repeating units also explain the previously observed cross-reactivity between the strains and to E. coli O164, which only differs in the terminal sugar residue that is lacking the (R)-1-carboxyethyl group.     

Structural determination of the O-antigenic polysaccharide from Escherichia coli O74

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O74 has been determined. Component analysis, together with ¹H and ¹³C NMR spectroscopy as well as ¹H,¹⁵N-HSQC experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure:

Full-size image (5K)

Cross-peaks of low intensity from an α-linked N-acetylglucosamine residue were present in the NMR spectra, and spectral analysis indicates that they originate from the penultimate residue in the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The ¹H, ¹³C and ¹⁵N NMR chemical shifts of the α- and β-anomeric forms of d-Fucp3NAc are also reported. The repeating unit of the E. coli O74 O-antigen is identical to that of the capsular polysaccharide from E. coli K45.     

Structural determination of the O-antigenic polysaccharide from the Shiga toxin-producing Escherichia coli O171

The structure of the O-antigenic part of the lipopolysaccharide (LPS) obtained from the verotoxin-producing Escherichia coli O171 has been determined. (1)H and (13)C NMR spectroscopy techniques in combination with component analysis were used to elucidate the O-antigen structure of O-deacylated LPS. Subsequent NMR analysis of the native LPS revealed acetylation at O-7/O-9 of the sialic acid residue. The sequence of sugars was determined by inter-residue correlations in (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation spectra. The O-antigen is composed of pentasaccharide repeating units with one equivalent of O-acetyl groups distributed over two positions: -->4)-alpha-Neu5Ac7,9Ac-(2-->6)-beta-D-Galp-(1-->6)-beta-DGlcp-->(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1--> Based on biosynthetic considerations, this should also be the biological repeating unit.     

Structural analysis of the O-antigen polysaccharide from Escherichia coli O152

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined. Component analysis together with 1H, 13C and 31P NMR spectroscopy were used to elucidate the structure. Inter-residue correlations were determined by 1H,31P COSY, 1H,1H NOESY and 1H,13C heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. The structure is similar to that of the O-antigen polysaccharide from E. coli O173. The cross-reactivity between E. coli O152 and E. coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

Structural elucidation of the O-antigenic polysaccharides from Escherichia coli O21 and the enteroaggregative Escherichia coli strain 105.

The structure of the O-antigen polysaccharide of the lipopolysaccharide from an enteroaggregative Escherichia coli (strain 105) has been elucidated, using primarily one-dimensional and two-dimensional NMR experiments. The sequence of residues was deduced with heteronuclear multiple-bond correlation and NOESY experiments. The structure of the repeating unit of the polysaccharide from the enteroaggregative E. coli is as follows:[sequence: see text] The structure of the O-antigen from enteroaggregative E. coli strain 105 was shown to be identical with that of E. coli O21 by sugar and methylation analyses as well as by 1H-NMR and 13C-NMR spectroscopy.     

Structural elucidation of the O-antigenic polysaccharide from enterohemorrhagic (EHEC) Escherichia coli O48:H21

The structure of the antigenic O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) produced by the enterohemorrhagic strain of Escherichia coli O48:H21 (EHEC) has been elucidated. The O-PS obtained by mild acid hydrolysis of the LPS had [alpha]D +95 (water) and was composed of L-rhamnose (L-Rha), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN), and D-galacturonic acid (D-GalA) (1:1:1:1:1). From the results of methylation analysis, mass spectrometry, 2D NMR, and DOC-PAGE, the O-PS was shown to be a high molecular mass polymer of a repeating pentasaccharide unit having the structure: [structure: see text]. The D-Gal pA non-reducing end groups in the O-PS were partially O-acetylated (approximately 30%) at the O-2 and O-3 positions and the degree of acetylation was variable from batch to batch cell production.     

Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O173

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O173 has been investigated. Sugar and methylation analyses, electrospray ionisation mass spectrometry together with ¹H, ³¹P and ¹³C NMR spectroscopy were the main methods used. The structure of the pentasaccharide repeating unit of the PS was found to be:

By treatment with 48% HF the phosphoric diester linkage was cleaved together with the glycosidic linkage of the fucosyl group, rendering a tetrasaccharide with the structure:

     

Synthesis of the 'primer-adaptor' trisaccharide moiety of Escherichia coli O8, O9, and O9a lipopolysaccharide

Described is the synthesis of the trisaccharide alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-beta-D-GlcpNAcO(CH2)8N3, the glycan portion of which corresponds to the 'adaptor-primer' moiety linking the O-chain and core oligosaccharide in the lipopolysaccharide of several Escherichia coli and Klebsiella pneumoniae serotypes. This report represents the first synthesis of this trisaccharide motif, and in the route involved, a key step is a [2+1] coupling of a protected Manp-(1-->3)-alpha-D-Manp glycosyl donor with a GlcpNAc acceptor. The azido group was included in the target to facilitate future preparation of neoglycoconjugates.     

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter baumannii strain 24

Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.     

Structural studies of the O-antigenic oligosaccharide from Vibrio salmonicida strain C2 isolated from Atlantic cod, Gadus morhua L

We report the chemical structure of the oligosaccharide part of Vibrio salmonicida lipopolysaccharide serotype C2, isolated from Atlantic cod (Gadus morhua L.). The structure was established by NMR spectroscopy and mass spectrometry. It is concluded that the oligosaccharide has the following structure in which L-alpha-D-Hepp is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hepp is D-glycero-alpha-D-manno-heptopyranose, alpha-NonA is 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-alpha-D-galacto-nonulosonic acid, and PEA is phosphoethanolamine. [chemical structure: see text]     

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains

Background

Enteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.

Results

The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage {"type":"entrez-protein","attrs":{"text":"P13374","term_id":"138227","term_text":"P13374"}}P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber.

Conclusions

At least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-574) contains supplementary material, which is available to authorized users.