The identity of the O-specific polysaccharide structure of Citrobacter strains from serogroups O2, O20 and O25 and immunochemical characterisation of C. youngae PCM 1507 (O2a,1b) and related strains

Serological studies using SDS-PAGE and immunoblotting revealed that from five strains that are ascribed to Citrobacter serogroup O2, four strains, PCM 1494, PCM 1495, PCM 1496 and PCM 1507, are reactive with specific anti-Citrobacter O2 serum. In contrast, strain PCM 1573 did not react with anti-Citrobacter O2 serum and, hence, does not belong to serogroup O2. The LPS of Citrobacter youngae O2a,1b (strain PCM 1507) was degraded under mild acidic conditions and the O-specific polysaccharide (OPS) released was isolated by gel chromatography. Sugar and methylation analyses along with (1)H- and (13)C-NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and (1)H,(13)C HSQC experiments, showed that the repeating unit of the OPS has the following structure: [structure: see text]. NMR spectroscopic studies demonstrated that Citrobacter werkmanii O20 and C. youngae O25 have the same OPS structure as C. youngae O2. Sugar and methylation analyses of the core oligosaccharide fractions demonstrated structural differences in the lipopolysaccharide core regions of these strains, which may substantiate their classification in different serogroups.     

Structural and serological studies on a new 4-deoxy-d-arabino-hexose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6).

The O-specific polysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) was isolated by mild acid hydrolysis of the lipopolysaccharide (LPS) and found to contain d-fucose, l-rhamnose, 4-deoxy-d-arabino-hexose and O-acetyl groups in molar ratios 2 : 1 : 1 : 1. On the basis of methylation analysis and 1H and 13C NMR spectroscopy data, the structure of the branched tetrasaccharide repeating unit of the O-specific polysaccharide was established. Using various serological assays, it was demonstrated that the LPS of strain PCM 1531 is not related serologically to other known 4-deoxy-d-arabino-hexose-containing LPS from Citrobacter PCM 1487 (serogroup O5) or C. youngae PCM 1488 (serogroup O36). Two other strains of Citrobacter, PCM 1504 and PCM 1505, which, together with strain PCM 1531, have been classified in serogroup O6, were shown to be serologically distinct from strain PCM 1531 and should be reclassified into another serogroup.     

Structures of two O-polysaccharides of the lipopolysaccharide of Citrobacter youngae PCM 1538 (serogroup O9)

Mild acid degradation of the lipopolysaccharide of Citrobacter youngae O9, strain PCM 1538 released a homopolysaccharide of 4-acetamido-4,6-dideoxy-D-mannose (D-Rha4NAc, N-acetyl-D-perosamine). Studies by methylation analysis and (1)H and (13)C NMR spectroscopy, using two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and H-detected (1)H,(13)C HSQC experiments showed the presence of two structurally different polysaccharides consisting of the following units: -->)-alpha-D-Rhap4NAc-(1 --> and --> 3)-alpha-D-Rhap4NAc-(1 --> 3)-beta-D-Rhap4NAc-(1 -->.     

Structure of the glycerol phosphate-containing O-specific polysaccharide and serological studies on the lipopolysaccharides of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14)

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to contain D-glucose, D-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a beta configuration, except for the side-chain Glc, which is alpha. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti-C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue.     

Structure of the O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii O11, strain PCM 1540

The O-specific polysaccharide of the lipopolysaccharide of Citrobacter gillenii PCM 1540 (serogroup O11) consists of D-Glc, D-Man, D-GalNAc, D-GlcNAc, 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) and O-acetyl groups in the ratios 2:1:1:1:1:1. On the basis of sugar and methylation analyses and Smith-degradation along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched hexasaccharide repeating unit was established: [structure: see text]. Citrobacter werkmanii PCM 1541 belonging to the same serogroup O11 was found to have an R-form lipopolysaccharide devoid of the O-specific polysaccharide.     

Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1546

An acidic O-polysaccharide isolated by mild acid hydrolysis from the lipopolysaccharide of Hafnia alvei PCM 1546 is composed of D-Gal, D-Glc, D-GlcA, D-GalNAc and O-acetyl groups in the ratios 1:1:1:2:1.6. On the basis of sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the pentasaccharide repeating unit of the polysaccharide was established: [see equation in text].     

New structure for the O-polysaccharide of Providencia alcalifaciens O27 and revised structure for the O-polysaccharide of Providencia stuartii O43

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O27 and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments. It was found that the polysaccharide is built up of linear partially O-acetylated tetrasaccharide repeating units and has the following structure: [structure: see text] where Qui4NFo stands for 4-formamido-4,6-dideoxyglucose (4-formamido-4-deoxyquinovose). The O-polysaccharide structure of Providencia stuartii O43 established earlier was revised with respect to the configuration of the constituent 4-amino-4,6-dideoxyhexose (from Rha4N to Qui4N).     

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O47

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O47:H4, strain 3646/51. Studies by sugar and methylation analyses along with Smith degradation and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY and H-detected 1H,13C HSQC and HMBC experiments, showed that the polysaccharide has a branched hexasaccharide repeating unit with the following structure: [carbohydrate structure: see text]     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O29

The O-polysaccharide was obtained by a mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O29. Structural studies were performed using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional 1H, 1H COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. On the basis of the data obtained, the following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: [structure: see text].     

Structure and cross-reactivity of the O-antigen of Providencia stuartii O18 containing 3-acetamido-3,6-dideoxy-D-glucose

The O-polysaccharide (O-antigen) of Providencia stuartii O18 was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text] where Qui3NAc is 3-acetamido-3,6-dideoxyglucose. Anti-P. stuartii O18 serum cross-reacted with the O-antigen of Proteus genomospecies 4, which could be accounted for the marked structural similarities of the main chain.     

The O-polysaccharide from the lipopolysaccharide of Providencia stuartii O44 contains L-quinovose, a 6-deoxy sugar rarely occurring in bacterial polysaccharides

The O-polysaccharide (O-antigen) of Providencia stuartii O44:H4 (strain 3768/51) was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC, and HMQC-TOCSY experiments. The O-polysaccharide was found to have a branched hexasaccharide repeating unit of the following structure: [Formula: see text].     

Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1529

The following structure of the pentasaccharide repeating unit of an acidic O-polysaccharide of Hafnia alvei PCM 1529 was established by sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy: [Carbohydrate structure: see text].     

Structure of the O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Providencia alcalifaciens O32 containing N-acetylisomuramic acid

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O32 and studied by sugar and methylation analyses, solvolysis with triflic acid, 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. It was found that the polysaccharide has a branched tetrasaccharide repeating unit containing 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (D-GlcNAc3Slac, N-acetylisomuramic acid) with the following structure: [STRUCTURE: SEE TEXT]. Serological studies with O-antisera showed antigenic relationships between P. alcalifaciens O32 and O29 as well as several other Providencia and Proteus strains sharing putative epitopes on the O-polysaccharides.     

Structure of a colitose-containing O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O6

The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O6 and studied by sugar and methylation analysis, selective hydrolytic removal of 3,6-dideoxy-L-xylo-hexose (colitose, Col), (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY and H-detected (1)H,(13)C HSQC and HMBC experiments. The polysaccharide was found to have a branched pentasaccharide repeating unit with the following structure: [see text] Remarkably, the trisaccharide side chain of the O6-polysaccharide represents a colitose ('3-deoxy-L-fucose') analogue of the H type 1 (precursor) antigenic determinant.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.     

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).     

Structure of the O-polysaccharide of Providencia stuartii O4 containing 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose

The O-polysaccharide of Providencia stuartii O4 was obtained by mild acid degradation of the lipopolysaccharide, and the following structure of the pentasaccharide repeating unit was established: [structure: see text] where D-Qui4N(L-AspAc) is 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose, which has not been hitherto found in bacterial polysaccharides. Structural studies were performed using sugar and methylation analyses, Smith degradation and NMR spectroscopy, including conventional 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments as well as COSY and NOESY experiments run in an H(2)O-D(2)O mixture to reveal correlations for NH protons.     

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O57 containing an amide of D-galacturonic acid with L-alanine

The O-polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O57:H29. Studies by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments, showed that the polysaccharide contains an amide of D-galacturonic acid with L-alanine and has the following pentasaccharide repeating unit: [formula: see text]     

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].     

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.