New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.     

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 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 a new 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii PCM 1542

The O-specific polysaccharide of Citrobacter gillenii PCM 1542 from serotype O-12a,12 b is composed of one residue each of D-glucose, D-GlcNAc, 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose (D-GlcNAcyl) and two GalNAc residues. On the basis of sugar and methylation analyses of the intact and Smith degraded polysaccharides, along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched pentasaccharide repeating unit of the O-specific polysaccharide was established:This structure differs significantly from that of the O-specific polysaccharide of C. gillenii PCM 1544 from the same serotype O-12a,12 b, which has been established earlier (Kübler-Kielz.shtsls;b, J. et al. Carbohydr. Res. 2001, 331, 331-336). Serological studies confirmed that the two O-antigens are not related and suggested that strains PCM 1542 and 1544 should be classified into different O-serogroups.     

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established: -->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Galp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-GlcpNAc-(1-->.     

Structures of the O-specific polysaccharides from Yokenella regensburgei (Koserella trabulsii) strains PCM 2476, 2477, 2478, and 2494: high-resolution magic-angle spinning NMR investigation of the O-specific polysaccharides in native lipopolysaccharides and directly on the surface of living bacteria.

The structures of the carbohydrate O-specific side-chain moiety of the lipopolysaccharides (LPS) of Yokenella regensburgei, strains PCM 2476, 2477, 2478, and 2494, have been investigated by (1)H and (13)C NMR, fast atom bombardment tandem mass spectrometry (FAB-MSMS), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, methylation analysis, partial acid hydrolysis, and immunological methods. It was concluded that the O-specific polysaccharides of strains 2476, 2477, 2478, and 2494 are composed of the same basic trisaccharide repeating unit having the structure -->3)-alpha-D-FucpNAc-(1-->2)-L-alpha-D-Hepp-(1-->3)-6-deoxy -alpha-L- Talp-(1-->, in which L-alpha-D-Hepp is L-glycero-alpha-D-manno-heptopyranose. The detailed analysis revealed, however, differences in O-acetylation patterns of the 6-deoxy-L-talose residue, with 2- and 4-O-acetyl disubstituted -->3)-6-deoxy-alpha-L-Talp-(1--> in strain PCM 2476 and a 2-O-acetylated residue in strains 2477, 2478, and 2494. These structures represent novel, trisaccharide repeating units of bacterial O-antigens that are characteristic and unique to the Y. regensburgeispecies. By use of the high-resolution magic-angle spinning (HR-MAS) technique, (1)H NMR spectra of the O-polysaccharides directly in isolated LPS were obtained. This allowed for almost full assignment and structural determination of the polysaccharide. By this technique the O-polysaccharide components were also observed in their original form directly on the surface of living bacterial cells.     

Identification of the methyl phosphate substituent at the non-reducing terminal mannose residue of the O-specific polysaccharides of Klebsiella pneumoniae O3, Hafnia alvei PCM 1223 and Escherichia coli O9/O9a LPS

O-specific polysaccharides of Gram-negative bacteria are synthesized by two different mechanisms: polymerization of the pre-formed O-repeating unit or sequential addition of the monosaccharides to the growing polysaccharide chain. In the second case, growth of the polymer can be further subdivided into two groups depending on the presence or absence of a special monosaccharide or non-sugar substituent that terminates the glycan. A family of polymannose O-polysaccharides provides prototypes for the chain terminating process. Polysaccharides of Klebsiella pneumoniae O3, Hafnia alvei PCM 1223, and Escherichia coli O9 have the same penta-mannose repeating unit. E. coli O9a has tetra-mannose repeat and this structure can be produced by mutants of E. coli O9. The mechanism of biosynthesis of H. alvei 1223 O-polysaccharide has not been reported. Here we show that all above polysaccharides contain the same modification at the non-reducing end; presence of a methyl phosphate group at O-3 of α-mannopyranose, that serves as the signal for termination of the chain elongation.     

Structures of the biological repeating units in the O-chain polysaccharides of Hafnia alvei strains having a typical lipopolysaccharide outer core region

Earlier, the structures of the O-chain polysaccharides of the lipopolysaccharides (LPS) of a number of Hafnia alvei strains have been established. However, it remained unknown, which is the first and the last monosaccharide of the O-chain. This is defined by the structure of the so-called biological repeating unit (O-unit), which is pre-assembled and then polymerised in the course of biosynthesis of bacterial polysaccharides by the Wzy-dependent pathway. Now we report on the structures of the O-units in 10 H. alvei strains. The LPS were cleaved by mild acid hydrolysis and oligosaccharide fractions IIIa and IIIb were isolated by gel chromatography subsequently on Sephadex G-50 and BioGel P-2 and studied by methylation analysis and NMR spectroscopy. Fraction IIIb was found to represent the core oligosaccharide containing a terminal upstream alpha-d-Glc-(1-->3)-alpha-d-Glc or alpha-d-Gal-(1-->3)-alpha-d-Glc disaccharide in the outer region that is typical of H. alvei. Fraction IIIa consists of the LPS core with one O-unit linked by a 3-substituted beta-d-GalNAc residue (in strains PCM 1189 and PCM 1546) or a 3-substituted beta-d-GlcNAc residue (in the other strains studied). In most strains examined the beta-configuration of the d-GlcNAc linkage in the first O-unit attached to the core is the same and in some strains is opposite to that found in the interior O-units of the O-chain polysaccharide. Various monosaccharides, including d-Glc, d-Gal, d-GlcA and acyl derivatives of 3-amino-3,6-dideoxy-d-glucose or 4-amino-4,6-dideoxy-d-glucose, occupy the non-reducing end of the O-unit.     

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.     

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.     

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 studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide.

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS, fast atom bombardment (FAB)-MS/MS and 1H- and 13C-NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: The polysaccharide is partially (approximately 10%) substituted with O-acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR-MAS) NMR analysis, which showed both the signals of the O-specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.     

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 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 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].     

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 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-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].     

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.     

Structure and serological analysis of the Hafnia alvei 481-L O-specific polysaccharide containing phosphate in the backbone chain

The lipopolysaccharide was extracted from cells of Hafnia alvei 481-L bacterial strain and, after mild acid hydrolysis, the O-specific polysaccharide was isolated and characterised. On the basis of chemical analyses and NMR spectroscopic studies of the polysaccharide and oligosaccharides obtained after Smith degradation, or hydrogen fluoride treatment, it was found that the repeating unit of the O-specific polysaccharide is a phosphorylated hexasaccharide: [see text]. The biological repeating unit of the H. alvei 481-L O-antigen has galactose phosphate at the nonreducing terminus. Serological tests indicate that this strain represents an individual serotype in the H. alvei genus.