Structural analysis of Edwardsiella tarda PCM 1155 O-polysaccharide revealed the presence of unique ß-L-RhapNAc3NAc derivative.

The chemical structure of the lipopolysaccharide O-polysaccharide repeating unit of Edwardsiella tarda strain PCM 1155 was studied for the first time. The complete structure of repeating unit was investigated by chemical methods, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The rarely occurring monosaccharide, 2,3-diacetamido-2,3,6-trideoxy-l-mannose (L-RhapNAc3NAc) was identified. The following structure was established.

Re-classification within the serogroups O3 and O8 of Citrobacter strains.

BACKGROUND: Citrobacter strains are opportunistic pathogens often responsible for serious enteric as well as extra-intestinal diseases, and therefore the O-antigenic scheme, still in use in diagnostic identification, should be set for proper serotyping. The structures of more than 30 different Citrobacter O-antigens (O-polysaccharide chains of the lipopolysaccharides) of 43 Citrobacter O-serogroups have been elucidated so far. However, relationships between strains in several heterogeneous serogroups still need to be clarified by immunochemical studies. These include complex serogroups O3 and O8, represented by 20 and 7 strains, respectively, which are the subject of the present work. Earlier, the O-polysaccharide structures have been determined for Citrobacter O3 strain Be35/57 (PCM 1508) and Citrobacter O8 strain Be64/57 (PCM 1536). RESULTS: Serological studies (immunoblotting) carried out on Citrobacter lipopolysaccharides from different strains ascribed to serogroups O3 and O8 showed that each of these serogroups should be divided into non-cross-reacting subgroups. Based on the results of chemical analyses and 1H and 13C NMR spectroscopy the structure of Citrobacter O-antigens from strains PCM 1504 (O6) and PCM 1573 (O2) have been established. Chemical data combined with serological analyses showed that several Citrobacter strains should be reclassified into other serogroups. CONCLUSIONS: Immunochemical studies carried out on Citrobacter LPS, described in this paper, showed the expediency of reclassification of: 1) strains PCM 1504 and PCM 1573 from serogroups O6 and O2 to serogroups O3 and O8, respectively, 2) strains PCM 1503 and PCM 1505 from serogroups O3 and O8 to new serogroups O3a and O8a, respectively.

Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells.

LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-ß1,3]GalNAc-α1,3-GalNAc-ß1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2.

Structure of the O-polysaccharide of Edwardsiella tarda PCM 1156.

Mild acid degradation of the lipopolysaccharide of Edwardsiella tarda PCM 1156 afforded an O-polysaccharide, which was isolated by gel-permeation chromatography on Sephadex G-50 and studied by sugar and methylation analyses along with (1)H NMR and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit of the O-polysaccharide was established: [structure: see text].

Studies on the O-antigen of Hafnia alvei PCM 1224 structurally and serologically related to the O-antigen of H. alvei 481-L.

Mild hydrolysis of the lipopolysaccharide of Hafnia alvei PCM 1224 at pH 4.2 cleaved partially the O-polysaccharide chain by the glycosyl phosphate linkages to yield a phosphorylated hexasaccharide representing the repeating unit of the O-polysaccharide and higher oligosaccharides consisting of two or more repeating units. Studies of the degradation products before and after dephosphorylation by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, HSQC-TOCSY, and (1)H,(31)P HMBC experiments, enabled elucidation of the following structure of the O-polysaccharide: [formula - see text]. This structure is similar to that of the O-polysaccharide of H. alvei 481-L established earlier (Kubler-Kielb, J.; Vinogradov, E.; Garcia Fernandez, J. M.; Szostko, B.; Zwiefka, A.; Gamian, A. Carbohydr. Res.2006, 341, 2980-2985), which has the same sugar composition and the same main chain structure and differs in the site of attachment of α-d-Glcp only. A two-way serological cross-reactivity of the lipopolysaccharides of H. alvei PCM 1224 and 481-L with polyclonal rabbit antisera indicated the expediency of classification of both strains to the same O-serogroup.

Structure of the O-polysaccharide of Edwardsiella tarda PCM 1150 containing an amide of D-glucuronic acid with L-alanine.

Mild acid degradation of the lipopolysaccharide of Edwardsiella tarda PCM 1150 afforded an O-polysaccharide, which was isolated by GPC on Sephadex G-50 and studied by sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopies, including experiments performed in a 9:1 H2O/D2O mixture to detect NH protons and their correlations with CH protons. The O-polysaccharide was found to contain an amide of d-glucuronic acid with l-alanine (d-GlcA6Ala) and the following structure of the branched hexasaccharide repeating unit was established: -->4)-ß-D-GlepA6Ala-(1-->4)-α-L-Fucp-(1-->4)-α-D-Glcp-(1-->4)-α-D-Quip-(1-->3)-ß-D-GlcpNAc-(1-->3<--1α-D-GalpNAc.

Structural and serological studies on the O-antigen show that Citrobacter youngae PCM1505 must be classified to a new Citrobacter O-serogroup.

The O-polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter youngae PCM1505 was studied by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [Formula: see text]. Structural and serological data obtained earlier and in this work show that the strain studied is a candidate to a new Citrobacter O-serogroup.

Structures of a unique O-polysaccharide of Edwardsiella tarda PCM 1153 containing an amide of galacturonic acid with 2-aminopropane-1,3-diol and an abequose-containing O-polysaccharide shared by E. tarda PCM 1145, PCM 1151 and PCM 1158.

Lipopolysaccharides of four strains of Edwardsiella tarda were degraded by mild acid hydrolysis, and the released O-polysaccharides were isolated by GPC 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, (1)H, (13)C HMBC, HSQC and HSQC-TOCSY experiments. The O-polysaccharide from E. tarda PCM 1153 was found to contain D-GalA, D-GlcNAc, D-Gal and 2-amino-1,3-propanediol (GroN). In the tetrasaccharide repeating unit, GroN is amide-linked to one of the GalA residues, and Gal is non-stoichiometrically 2- or 3-O-acetylated (~45% at each position): [structure: see text]. Three other E. tarda strains examined (PCM 1145, PCM 1151 and PCM 1158) share the following O-polysaccharide structure: [structure: see text] where Abe indicates 3,6-dideoxy-D-xylo-hexose (abequose). This structure resembles those of Citrobacter freundii O22 (PCM 1555) and Salmonella enterica O4. In accordance with the structural data, SDS-PAGE and immunoblotting of the lipopolysaccharides with anti-C. freundii O22 serum demonstrated that the O-antigens of the three E. tarda strains are serologically identical to each other and to the O-antigens of C. freundii O22 and S. enterica O4.

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.

Structure of the O-polysaccharide of Citrobacter youngae PCM 1503.

The O-polysaccharide (O-antigen) was obtained from the lipopolysaccharide of Citrobacter youngae PCM 1503, which is currently classified to the O7 serogroup. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy: Structural and serological data of the O-antigen suggest that strain PCM 1503 should be reclassified to a new Citrobacter serogroup.

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 (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H ROESY and (1)H,(13)C HMBC experiments, showed that the repeating unit of the O-polysaccharide has the following structure: alpha-Abep 1 -->3 --> 2)-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Galp-(1--> 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.

Immunochemical studies of the lipopolysaccharides of Hafnia alvei PCM 1219 and other strains with the O-antigens containing D-glucose 1-phosphate and 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose.

INTRODUCTION: Hafnia alveiis the only species of the genus Hafnia, which belongs to the family of Enterobacteriaceae. These Gram-negative bacteria are commonly distributed in the natural environment and are often the cause of human opportunistic infections. Their lipopolysaccharides (LPSs) are important surface antigens which are responsible for the serological specificity and numerous cross-reactions with other enterobacterial genera. So far, 29 different O-polysaccharide (OPS, O-antigen) structures in Hafnias LPSs have been established and for some of them the molecular basis of the serological activity has been elucidated. MATERIALS AND METHODS: OPS from H. alvei strain PCM 1219 was obtained by mild acid hydrolysis of the LPS followed by gel permeation chromatography of carbohydrate material on Sephadex G-50 column. The polysaccharide structure was determined using chemical methods as well as (13)C NMR and (1)H NMR spectroscopy. For serological studies, SDS-PAGE, immunoblotting, and passive hemagglutination tests were used. RESULTS: The serological studies revealed a cross-reactivity of the LPSs of H. alvei PCM 1219 and a group of H. alvei strains with an O-antigen containing D-glucose 1-phosphate and [(R)-3-hydroxybutyramido]-D-glucose. The following structure of the OPS was established: where Acyl stands for (R)-3-hydroxybutyryl and the degree of O-acetylation is ~70%. The structure of the core oligosaccharide was found to be typical of the genus Hafnia. CONCLUSIONS: Based on the OPS structure and serological results it was concluded that H. alvei strain PCM 1219 should be classified in the same serogroup as the H. alvei type strain ATCC 13337 and five other strains containing D-glucose 1-phosphate and 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose in their O-antigens.

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 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.

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-polysaccharide of Hafnia alvei strain PCM 1189 that has hexa- to octasaccharide repeating units owing to incomplete glucosylation.

The O-polysaccharide of Hafnia alvei PCM 1189 consists of D-glucose, D-galactose, D-GalNAc and D-GlcA and lacks the strict regularity. The intact and carboxyl-reduced polysaccharides as well as oligosaccharides obtained by partial acid hydrolysis were studied by chemical and enzymatic analyses, methylation and NMR spectroscopy. The following structure was established for the O-polysaccharide, which is built up of branched hexa- to octasaccharide repeating units differing in the number of lateral glucose residues: [structure: see text] where the glucose residues shown in italics are nonstoichiometric substituents. The repeating units include also a minor O-acetyl group, whose position was not determined.

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 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].