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-polysaccharide of Citrobacter youngae O1 containing an alpha-D-ribofuranosyl group

The lipopolysaccharide of Citrobacter youngae O1, strain PCM 1492 was degraded with acid or alkali under mild conditions, and the resultant polysaccharide was isolated by GPC and studied by sugar and methylation analyses and 1H and 13C NMR spectroscopies, including 2D COSY, TOCSY, NOESY and 1H, 13C HSQC experiments. The following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: [structure: see text] where substitution with the alpha-D-Ribf group is nonstoichiometric. This group occurs rarely in bacterial polysaccharides and is easily cleaved under mild acidic conditions. Studies with polyclonal rabbit antisera against whole cells of C. youngae PCM 1492 and PCM 1506 showed the serological identity of the lipopolysaccharides of C. youngae PCM 1492, PCM 1493 and PCM 1506, which are classified in serogroup O1.     

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.     

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 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 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.     

Structure of the O-specific polysaccharide of Vibrio cholerae O9 containing 2-acetamido-2-deoxy-D-galacturonic acid

The O-specific polysaccharide (OPS) was isolated by mild-acid degradation of the lipopolysaccharide of Vibrio cholerae O9 and studied by carboxyl reduction, sugar and methylation analyses, Smith degradation, and two-dimensional NMR spectroscopy, including COSY, TOCSY, NOESY, and H-detected 1H,(13)C HMQC experiments. The following structure of the pentasaccharide-repeating unit of the OPS was established:     

Structure of the O polysaccharide and serological classification of Pseudomonas syringae pv. ribicola NCPPB 1010.

The O polysaccharide (OPS) moiety of the lipopolysaccharide (LPS) of a phytopathogenic bacterium Pseudomonas syringae pv. ribicola NCPPB 1010 was studied by sugar and methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy, including 2D COSY, TOCSY, NOESY and H-detected 1H,13C HMQC experiments. The OPS structure was elucidated, and shown to be composed of branched pentasaccharide repeating units (O repeats) of two types, major (1) and minor (2), differing in the position of substitution of one of the rhamnose residues. Both O repeats form structurally homogeneous blocks within the same polysaccharide molecule. Although P. syringae pv. ribicola NCPPB 1010 demonstrates genetic relatedness and similarity in the OPS chemical structure to some other P. syringae pathovars, it did not cross-react with any OPS-specific mAbs produced against heterologous P. syringae strains. Therefore, we propose to classify P. syringae pv. ribicola NCPPB 1010 in a new serogroup, O8.     

Structure of the O-polysaccharide leads to classification of Proteus penneri 31 in Proteus serogroup O19

O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C and 1H,31P HMQC experiments, demonstrated the following structure of the polysaccharide: [carbohydrate structure: see text] where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and alpha-L-FucNAc-(1-->3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31.     

Structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: (struture: see text). The main D-mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.     

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.     

Structure of a phosphoethanolamine-containing O-polysaccharide of Citrobacter freundii strain PCM 1443 from serogroup O39 and its relatedness to the Klebsiella pneumoniae O1 polysaccharide

Lipopolysaccharide was extracted from cells of Citrobacter freundii PCM 1443 from serogroup O39 and degraded by mild acid hydrolysis to give an O-polysaccharide. Based on enzymatic and methylation analyses, along with 1H and 13C nuclear magnetic resonance spectroscopy, it was found that the lipopolysaccharide studied has two different linear polysaccharide chains of d-galactan type containing 3-substituted galactose residues. One of the galactans has the disaccharide repeating units of alpha-D-galactopyranose and beta-D-galactofuranose and the other is comprised of alpha-D-galactopyranose and beta-D-galactopyranose, the latter being substituted in 25% repeats with PEtN at O-6. An immunoblotting assay demonstrated that the lipopolysaccharide of C. freundii PCM 1443 is serologically related to that of Klebsiella pneumoniae O1, which contains the same galactan chains but is devoid of phosphoethanolamine.     

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]     

Serotyping of clinical isolates belonging to Proteus mirabilis serogroup O36 and structural elucidation of the O36-antigen polysaccharide

The O-specific polysaccharide (OPS) isolated from the lipopolysaccharide of Proteus mirabilis O36 was found to have a pentasaccharide repeating unit of the following structure: -->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAc6Ac-(1-->4)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. The structure is unique among Proteus OPS, which is in agreement with the classification of this strain into a separate Proteus O-serogroup. Remarkably, the P. mirabilis O36-polysaccharide has the same structure as the OPS of Escherichia coli O153, except that the latter is devoid of O-acetyl groups. The cross-reaction of anti-O36 antibodies with the O-part of E. coli O153 lipopolysaccharide is observed. In the present study, two steps of serotyping Proteus strains are proposed: screening of dry mass with enzyme-linked immunosorbent assay and immunoblot with the crude lipopolysaccharides. This method allowed serotyping of 99 P. mirabilis strains infecting the human urinary tract. Three strains were classified into serogroup O36. The migration pattern of these lipopolysaccharides fraction with long O-specific PSs was similar to the standard laboratory P. mirabilis O36 (Prk 62/57) lipopolysaccharide. The relatively low number of clinical strains belonging to serogroup O36 did not correspond to the presence of anti-P. mirabilis O36 antibodies in the blood donors' sera. Twenty-five percent of tested sera contained a statistically significant elevated level of antibodies reacting with thermostable surface antigens of P. mirabilis O36. The presence and amount of antibodies correlated with Thr399Ile TLR4 polymorphism types (P=0.044).     

Structural studies of glucorhamnans isolated from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O4 and O7, and of an O14 strain

Partially acetylated glucorhamnans have been isolated from the lipopolysaccharides of three strains of Serratia marcescens. The polymer from the reference strain (C.D.C. 864-57) for serogroup O4 has the disaccharide repeating-unit shown below, in which acetylation at position 2 of the rhamnosyl residue is approximately 90% complete. Similar glucorhamnans from the reference strain (C.D.C. 843-57) for serogroup O7 and from a pigmented strain (NM) of serogroup O14 differ only in the configuration of the L-rhamnopyranosyl residue (beta) and the extent of O-acetylation (O7, almost stoichiometric; NM, 80-90%). Glucorhamnans of the second type have been isolated previously from the lipopolysaccharides of other strains of S. marcescens, including the reference strain for serogroup O6 and another pigmented O14 strain (N.C.T.C. 1377). In all cases, the lipopolysaccharide extracts also contained acidic glycans, but the glucorhamnans are believed to constitute the integral side-chains. (Formula: see text).     

Structure of an abequose-containing O-polysaccharide from Citrobacter freundii O22 strain PCM 1555

The lipopolysaccharide of Citrobacter freundii O22 (strain PCM 1555) was degraded under mild acidic conditions and the O-polysaccharide released was isolated by gel chromatography. Sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ¹H,¹H ROESY and ¹H,¹³C HMBC experiments, showed that the repeating unit of the O-polysaccharide has the following structure:

Full-size image (4K)

where Abe is abequose (3,6-dideoxy-d-xylo-hexose). SDS–PAGE and immunoblotting revealed that the O-antigen of C. freundii O22 is serologically indistinguishable from those of Salmonella group B serovars (Typhimurium, Brandenburg, Sandiego, Paratyphi B) but not related to other abequose-containing O-antigens tested (Citrobacter werkmanii O38 and Salmonella Kentucky) or colitose (l enantiomer of abequose)-containing O-antigen of Escherichia coli O111.     

The structure of the lipopolysaccharide core region of Citrobacter 027

The structure of Citrobacter 027 lipopolysaccharide core has been established using sugar and methylation analyses and 1H-NMR spectroscopy, and was shown to be identical to the core described recently in PCM 1487 strain which represents a separate serotype in Citrobacter genus.     

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.     

Structure and serologic properties of O-specific polysaccharide from Citrobacter freundii possessing cross-reactivity with Escherichia coli O157:H7

Citrobacter freundii OCU158 is a serologically cross-reactive strain with Escherichia coli O157:H7. To explore the close relationship between two strains, we have analyzed the chemical structures of O-specific polysaccharides and antigenic properties of lipopolysaccharides (LPSs) of both strains. The structure of O-specific polysaccharides from both strains was found to be identical by chemical and nuclear magnetic resonance analyses, in which D-PerNAc was 4-acetamido-4,6-dideoxy-D-mannose: [-->4)-beta-D-Glc-(1-->3)-alpha-D-PerNAc-(1-->4)-alpha-D-GalNAc-(1 --> 3)-alpha-L-Fuc-(1-->](n). The enzyme immunoassay using LPS derived either from E. coli O157 or from C. freundii could equally detect high levels of serum antibodies against LPS in patients with enterohemorrhagic E. coli (EHEC) O157 infection. Absorption of antibodies in EHEC patient serum by LPS from E. coli O157 or C. freundii, however, showed a difference in the epitopes. This difference was attributable to the epitope specificity of the core region and/or lipid A structure in LPS.