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-specific polysaccharide of Proteus vulgaris O4 containing a new component of bacterial polysaccharides, 4,6-dideoxy-4

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1 --> 3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-[N-[(R)-3-hydroxybutyryl]-L-alanyl]amino-D-glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: --> 4)-beta-D-GlcpA-(1 --> 3)-beta-D-GlcpNAc-(1 --> 2)-beta-D-Quip4N(HbAla)-(1 --> 3)-alpha-D-Galp-(1 -->. This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.     

The structure of Proteus mirabilis O3 O-specific polysaccharide containing N-(2-hydroxyethyl)-D-alanine

O-Specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O3 lipopolysaccharide. The polysaccharide was dephosphorylated with 48% HF to give a linear polysaccharide and an amino acid, N-(2-hydroxyethyl)-D-alanine. The structure of the polysaccharide was determined by methylation, Smith degradation and computer-assisted analysis of the 13C-NMR spectra of original and dephosphorylated polymers and oligomers. The structure of the amino acid was investigated by using 1H and 13C-NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative). Its absolute configuration was established by comparison of the optical rotation value and CD spectrum of the natural and synthetic product. On the basis of the data obtained, it was concluded that the repeating unit of P. mirabilis O3 O-specific polysaccharide has the following structure: (formula; see text) Removal of the amino acid phosphate substituent significantly decreased serological activity of the O-specific polysaccharide, thus showing the immunodominant role of this group. Serological cross-reactions between P. mirabilis O3 and O27 were demonstrated and tentatively substantiated.     

The structure of O-specific polysaccharide chains of lipopolysaccharides from Citrobacter 032 and Salmonella arizonae 064 (Arizona 29)

On the basis of acid hydrolysis, methylation, Smith degradation, selective cleavage with anhydrous hydrogen fluoride, and 13C NMR analysis, the repeating unit of the O-specific polysaccharide of Citrobacter O32 was concluded to have the following structure: (Formula: see text). The repeating unit of the Salmonella arizonae O64 O-specific polysaccharide has the same structure lacking the O-acetyl group.     

Antigenic bacterial polysaccharides. 24. The structure of the O-specific polysaccharide chain of Salmonella arizonae 063 (Arizona 08) lipopolysaccharide

The O-specific polysaccharide chain of the Salmonella arizonae O63 lipopolysaccharide is composed of D-glucose, D-galactose, N-acetyl-D-galactosamine, and 3-acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) residues in the ratio 1:1:2:1. On the basis of methylation analysis and calculations of 13C-NMR-spectra of the polysaccharide and of the product of its selective cleavage with anhydrous hydrogen fluoride, the linear polymer lacking 3-acetamido-3,6-dideoxygalactose, it was concluded that the polysaccharide has the following structure: (Formula: see text).     

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 63 from a new serogroup O68.

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O-specific polysaccharide that was isolated by gel-permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two-dimensional 1H, 1H COSY, TOCSY rotating-frame NOE spectroscopy, H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), and 1H, 13C HMQC-TOCSY experiments, demonstrated the following structure of the polysaccharide: where FucNAc is 2-acetamido-2,6-dideoxygalactose and PEtn is 2-aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of D-GlcNAc6PEtn and an alpha-L-FucNAc-(1-->3)-D-GlcNAc disaccharide, with other Proteus O-specific polysaccharides. A marked cross-reactivity of P. penneri 63 O-antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O-specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O-antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.     

Structure of the acidic O-specific polysaccharide from Proteus vulgaris O39 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid.

The O-specific polysaccharide of Proteus vulgaris O39 was found to contain a new acidic component of Proteus lipopolysaccharides, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac). The following structure of the polysaccharide was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with selective cleavage of the polysaccharide by solvolysis with anhydrous trifluoromethanesulfonic (triflic) acid: -->8)-beta-Psep5Ac7Ac-(2-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1--> The structure established is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied into a separate Proteus serogroup.     

Structure of the O-specific polysaccharide of Proteus mirabilis O11, another Proteus O-antigen containing an amide of D-galacturonic acid with L-threonine

The O-specific polysaccharide of Proteus mirabilis O11 was studied by sugar analysis, Smith degradation, 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and 1H-detected 1H, 13C HMQC experiments. The following structure of a pentasaccharide repeating unit of the polysaccharide was established: [formual: see text] where D-GalA6LThr is N-(D-galacturonoyl)-L-threonine. ELISA with anti-P. mirabilis O11 serum showed that D-GalA6LThr is of minor importance for manifesting the O11 immunospecificity.     

Structure of the O-specific polysaccharide from a marine bacterium Oceanisphaeralitoralis KMM 3654(T) containing ManNAcA

The O-specific polysaccharide was isolated from the lipopolysaccharide of a marine bacterium Oceanisphaeralitoralis KMM 3654(T) and studied by chemical methods along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-specific polysaccharide of O. litoralis containing D-glucose and two residues of 2-acetamido-2-deoxy-D-mannuronic acid was established: →4)-α-D-Glcp-(1→4)-β-D-ManpNAcA-(1→4)-β-D-ManpNAcA-(1→.     

Antigenic bacterial polysaccharides. 23. The structure of the O-specific polysaccharide chain of Salmonella arizonae 059 lipopolysaccharide

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.     

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 8 from a new serogroup O67.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Proteus penneri 8 lipopolysaccharide and found to contain D-glucose, D-galacturonic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2,6-dideoxy-L-galactose (L-FucNAc) and 2-aminoethyl phosphate (PEtn) in the ratios 2 : 1 : 1 : 1 : 1 : 1. 1H and 13C NMR spectroscopy was applied to the intact and dephosphorylated polysaccharides, and the following structure of the hexasaccharide repeating unit was established: The O-specific polysaccharide has a unique structure, and, accordingly, we propose for P. penneri 8 a new Proteus O67 serogroup, in which this strain is at present the single representative. The nature of epitopes on LPS of P. penneri 34, P. mirabilis O16, P. mirabilis O23 and P. vulgaris O22, which cross-react with O-antiserum against P. penneri 8, is discussed.     

Structure of the O-polysaccharide of Providencia stuartii O49

The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->     

Structure of the O-specific polysaccharide of Proteus penneri 71 and classification of cross-reactive P. penneri strains to a new proposed serogroup O64.

A neutral O-specific polysaccharide (O-antigen) was isolated from the lipopolysaccharide (LPS) of the bacterium Proteus penneri 71. On the basis of sugar analysis and 1H- and 13C-NMR spectroscopic studies, including two-dimensional COSY, 13C,1H heteronuclear COSY and ROESY, the following structure of the trisaccharide repeating unit of the polysaccharide was established: -->3)-beta-D-GlcpNAc-(1-->4)-beta-D-GlcpNAc-(1-->3)-alpha-D-Galp-(1-- > The polysaccharide has the same carbohydrate backbone as the O-specific polysaccharide of P. penneri 19 and both are similar to that of P. penneri 62 studied by us previously. A cross-reactivity of anti-P. penneri 71, 19 and 62 O-antisera with 11 P. penneri strains was revealed and substantiated at the level of the O-antigen structures. These strains could be divided into three subgroups within a new proposed Proteus O64 serogroup containing P. penneri strains only.     

Partial deletion of the cloned rfb gene of Escherichia coli O9 results in synthesis of a new O-antigenic lipopolysaccharide.

The rfb gene, involved in the synthesis of the O-specific polysaccharide (a mannose homopolymer) of Escherichia coli O9 lipopolysaccharide (LPS), was cloned in E. coli K-12 strains. The O9-specific polysaccharide covalently linked to the R core of K-12 was extracted from the K-12 strains harboring the O9 rfb gene. All the other genes required for the synthesis of rfe-dependent LPS are therefore considered to be present in the K-12 strains. It was found that bacteria harboring some clones with deletions of the ca. 20-kilobase-pair (kbp) BglII-StuI fragment no longer synthesized the O9-specific polysaccharide. However, bacteria harboring clones del 21, del 22, and del 25, which carry deletions of the 10-kbp PstI-StuI fragment, synthesized an O-specific polysaccharide antigenically distinct from E. coli O9 LPS. Although this new O-specific polysaccharide consisted solely of mannose and the mannose residues were combined only through alpha-1,2 linkage, it was still composed of a repeating oligosaccharide unit, possibly a trisaccharide unit,----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----. It is therefore likely that this new O-specific polysaccharide was derived from a part of the O9-specific polysaccharide----3)alpha Man-(1----3)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----and that the deleted part of the clones was responsible for the synthesis of alpha-1,3 linkages of the O9-specific polysaccharide.     

Structure of the O-specific polysaccharide isolated from the lipopolysaccharide of Citrobacter gillenii serotype O12a, 12b strain PCM 1544

A neutral O-specific polysaccharide was isolated from the lipopolysaccharide of Citrobacter gillenii strain PCM 1544, representing serotype O12a,12b. The polysaccharide was studied by sugar and methylation analyses and Smith degradation along with 1H and 13C NMR spectroscopy, including a ROESY experiment. The following structure of the tetrasaccharide repeating unit was established, in which substitution with terminal GlcNAc is approximately 60%. [structure: see text]     

Structural studies of the side chain of outer membrane lipopolysaccharide from Pseudomonas syringae pv. coriandricola W-43.

The lipopolysaccharide (LPS) was isolated from Pseudomonas syringae pv. coriandricola W-43 by hot phenol-water extraction. Rhamnose and 3-N-acetyl-3-deoxyfucose were found to be the major sugar constituents of the LPS together with N-acetylglucosamine, N-acetylgalactosamine, heptose, and 3-deoxy-D-manno-octulosonic acid (Kdo). The main fatty acids of lipid A of the LPS were 3-OH-C:10, C12:0, 2-OH-C12:0, and 3-OH-C12:0. The O-specific polysaccharide liberated from the LPS by mild-acid hydrolysis was purified by gel permeation chromatography. The compositional analysis of the O-specific polysaccharide revealed the presence of L-rhamnose and 3-N-acetyl-3-deoxy-D-fucose in a molar ratio of 4:1. The primary structure of the O-specific polysaccharide was established by methylation analysis together with 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional shift-correlated and one-dimensional nuclear Overhauser effect spectroscopy. The polysaccharide moiety was found to consist of a tetrasaccharide rhamnan backbone, and 3-N-acetyl-3-deoxy-D-fucose constitutes the side chain of the branched pentasaccharide repeating unit of the polysaccharide.     

Structure and cross-reactivity of the O-antigen of Proteus vulgaris O8.

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O8 lipopolysaccharide followed by gel permeation chromatography. Studies of the polysaccharide by sugar and methylation analyses and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, demonstrated the presence of a tetrasaccharide repeating unit having the following structure: [sequence: see text] The role of an epitope associated with the alpha-L-FucpNAc-(1-->3)-D-GlcpNAc disaccharide in serological cross-reactivity of P. vulgaris O8 is discussed.     

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.     

Structure of the O-specific polysaccharide of the bacterium Proteus mirabilis O29

An O-specific polysaccharide was obtained by mild acid degradation of P. mirabilis O29 lipopolysaccharide (LPS) and found to contain 2-acetamido-2-deoxy-D-galactose and D-glucuronic acid (D-GlcA) in the ratio 3:1. Studies of the polysaccharide by 1H- and 13C-NMR spectroscopy including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and H-detected 1H,13C-heteronuclear multiple-quantum coherence (HMQC) experiments demonstrated the following structure of the branched tetrasaccharide repeating unit: