Structure of the capsular polysaccharides and lipopolysaccharides from Haemophilus parasuis strains ER-6P (serovar 15) and Nagasaki (serovar 5).

Haemophilus parasuis is a Gram-negative bacterium from the family Pasteurellaceae and a swine pathogen. H. parasuis is found in the upper respiratory tract of piglets and produces Glässer's disease, an invasive disease characterized by polyserositis. H. parasuis contains a short lipopolysaccharide (LPS) or lipooligosaccharide (LOS) reported to play a partial role in interaction with host cells. The presence of capsule has been phenotypically demonstrated in certain H. parasuis strains and its role in virulence has been suggested, but the chemical structure of the surface polysaccharides of this bacterium was unknown. The structure of capsular polysaccharide (CPS) and LOS from virulent strains ER-6P and Nagasaki was studied by NMR spectroscopy, mass spectrometry and chemical methods. CPS from both strains had the same main chain with disaccharide repeating unit, substituted with α-Neu5R-(2-3)-α-GalNAc-(1-P-(strain ER-6P) or α-Neu5R-(2-3)-α-Gal-(1-P-strain Nagasaki) side chains, where R is the N-acetyl or N-glycolyl group. Glycolyl-neuraminic acid is widely found in animal glycoproteins, but it apparently has not been found in bacteria before, and might be important for the biology of this microorganism. Ac and Gc were present in equal amounts in the strain ER-6P but Nagasaki contained only about 20% of Gc substituent. Both strains produced the same LPS of a rough type with a single phosphorylated Kdo linking core and lipid A parts. LOS structure was similar to some strains of H. influenzae and contained a globotetraose terminal sequence.

Structure of the O-antigen polysaccharide present in the lipopolysaccharide of Cronobacter dublinensis (subspecies lactaridi or lausannensis) HPB 3169.

Cronobacter dublinensis (formerly Enterobacter sakazakii) HPB 3169 is a pathogenic Gram-negative bacterium that produces a smooth-type lipopolysaccharide in which the antigenic O-polysaccharide component was determined to be a repeating pentasaccharide unit composed of L-rhamnose; 2-acetamido-2-deoxy-D-glucose; 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-glucose; and 3-deoxy-manno-oct-2-ulosonic acid in the respective molar ratio 2:1:1:1. Chemical and 2D NMR analyses of the O-polysaccharide and a pentasaccharide derived by the mild acid hydrolysis of the ketosyl linkage of the Kdo (3-deoxy-D-manno-2-octulosonic acid) residue in the O-polysaccharide established that the O-antigen is a high molecular mass unbranched polymer of a repeating pentasaccharide unit and has the structure [see formula in text] where Bu is a (R)-3-hydroxybutanoyl substituent. The O-antigen is structurally similar to that of the recently reported Cronobacter sakazakii strain G706 (designated as serotype O5), except that in strain G706 the d-Qui3N is in its N-acetyl form, in contrast to its presence as a 3-deoxy-3-(R)-3-hydroxybutyramido derivative in the C. sakazakii HPB 3169 strain O-antigen.

An atypical biotype I Actinobacillus pleuropneumoniae serotype 13 is present in North America.

Atypical Actinobacillus pleuropneumoniae serotype 13 strains present in North America are described here for the first time. Different from serotype 13 strains described in Europe, North America strains are biotype I and antigenically related to both, serotypes 13 and 10. Chemical and structural analysis of the capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of a representative strain revealed that the CPS is almost identical to that of the reference strain of serotype 13, having a slightly higher degree of glycose O-acetylation. However, it produces an O-PS within the LPS antigenically and structurally identical with that of the reference strain of A. pleuropneumoniae serotype 10. The O-PS was characterized as a homopolymer of 1,2 linked ß-D-galactofuranosyl residues, a structure unrelated to that of the O-PS produced by the reference strain of serotype 13. Strains from Canada and United States are antigenically, phenotypically and genotypically similar. Animals infected by one of these strains induced antibodies that were detected by a LPS-based ELISA diagnostic test using either the homologous antigen or that of serotype 10. Based on the LPS and toxin profile, these strains might be misidentified as A. pleuropneumoniae serotype 10.

Characterization of the lipopolysaccharide O-antigen of Cronobacter turicensis HPB3287 as a polysaccharide containing a 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid) residue.

Cronobacter turicensis, previously known as Enterobacter sakazakii, is a Gram-negative opportunistic food-borne pathogen that has been reported as a cause of life-threatening neonatal infections. From chemical and physical analyses involving composition analysis, methylation, two-dimensional high-resolution nuclear magnetic resonance, and mass spectrometry methods, the antigenic O-polysaccharide in the smooth-type lipopolysaccharide of C. turicensis (strain HPB 3287) was determined to be a high molecular mass polymer of a repeating pentasaccharide unit composed of D-galactose, D-glucose, 2-acetamido-2-deoxy-D-galactose, and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid), in a molar ratio 2:1:1:1, and having the structure: [see formula in text].

Structural characterization and MHCII-dependent immunological properties of the zwitterionic O-chain antigen of Morganella morganii.

Morganella morganii is a commensal Gram-negative bacterium that has long been known to produce an antigen bearing phosphocholine groups. We determined the structure of this O-chain antigen and found that its repeating unit also contains a free amino group and a second phosphate: This alternating charge character places the M. morganii O-chain polysaccharide into a small family of zwitterionic polysaccharides (ZPSs) known to induce T-cell-dependent immune responses via presentation by class II major histocompatibility complex (MHCII) molecules. In vitro binding assays demonstrate that this O-chain interacts with MHCII in a manner that competes with binding of the prototypical ZPS antigen PSA from Bacteroides fragilis, despite its lack of a helical structure. Cellular studies also showed that the M. morganii polysaccharide induces activation of CD4(+) T-cells. Antibody binding experiments using acid hydrolyzed fragments representing the monomer and higher oligomers of the repeating unit showed that the phosphocholine group was the dominant element of the epitope with an overall affinity (K(D)) of about 5 × 10(-5) M, a typical value for an IgM anti-carbohydrate antibody but much lower than the affinity for phosphocholine itself. These data show that the structure of the M. morganii polysaccharide contains a unique zwitterionic repeating unit which allows for immune recognition by T-cells, making it the first identified T-cell-dependent O-chain antigen.

The structural characterization of the O-polysaccharide antigen of the lipopolysaccharide of Escherichiacoli serotype O118 and its relation to the O-antigens of Escherichiacoli O151 and Salmonellaenterica O47.

Mild acid hydrolysis of the lipopolysaccharide produced by Escherichiacoli O118:H16 standard strain (NRCC 6613) afforded an O-polysaccharide (O-PS) composed of d-galactose, 2-acetamidoylamino-2,6-dideoxy-L-galactose, 2-acetamido-2-deoxy-D-glucose, ribitol, and phosphate (1:1:1:1:1). From DOC-PAGE, sugar and methylation analyses, one- and two-dimensional NMR spectroscopy, capillary electrophoresis-mass spectrometry, hydrolysis, and sequential Smith-type periodate oxidation studies, the O-PS was determined to be an unbranched linear polymer having the structure: [6)-α-d-Galp-(1→3)-α-L-FucpNAm-(1→3)-ß-D-GlcpNAc-(1→3)-Rib-ol-5-P-(O→](n) The structure of the O-PS is consistent with the reported DNA data on the O-antigen gene-cluster of E. coli O118 and interestingly, the O-PS is similar to the structures of the O-antigens of Salmonellaenterica O47 and E. coli O151:H10 reference strain 880-67, as predicted from the results of DNA sequencing of their respective O-antigen gene-clusters.

The structure of the O-antigen of Cronobacter sakazakii HPB 2855 isolate involved in a neonatal infection.

Strains of Cronobactersakazakii (previously known as Enterobactersakazakii) are medically recognized important Gram-negative bacterial pathogens that cause enterocolitis, septicemia, and meningitis, with a high mortality rate in neonates. The structure of their O-antigens, that form part of their somatic lipopolysaccharide (LPS) components, is of interest for their chemical and serological identification and their relationship to virulence. The O-polysaccharide (O-PS) of C.sakazakii HPB 2855 (SK 81), a strain isolated from an infant at the Hospital for Sick Children in Toronto in 1981, was shown to be a polymer of a partially O-acetylated-repeating hexasaccharide unit composed of d-glucose, d-galacturonic acid, 2-acetamido-2-deoxy-d-galactose, and l-rhamnose (1:1:1:3). From composition and methylation analysis, and the application of 1D and 2D (1)H and (13)C NMR spectroscopy, the O-PS was determined to be a polymer of a repeating oligosaccharide unit having the structure:

The structure of the antigenic O-polysaccharide in the lipopolysaccharide of enterohaemorrhagic Escherichia coli serotype O71:H12.

The antigenic O-polysaccharide component of the lipopolysaccharide produced by Escherichia coli serotype O71:H12 was analyzed by chemical composition, nuclear magnetic spectroscopy, and Smith-type periodate oxidation methods. It was determined to be a partially O-acetylated unbranched polymer of a repeating tetrasaccharide unit composed of L-rhamnose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and 3-acetamido-3-deoxy-D-quinovose (1:1:1:1) residues having the following structure: [structure: see text]

Structural determination of the O-antigenic polysaccharide of enteropathogenic Escherichia coli O103:H2.

The structure of the antigenic O-polysaccharide isolated from the lipopolysaccharide produced by enterohemorrhagic Escherichia coli O103:H2 was determined and shown to be composed of d-glucose (1 part), 2-acetamido-2-deoxy-d-glucose (2 parts), 2-acetamido-2-deoxy-d-galactose (1 part), and 3-deoxy-3-(R)-3-hydroxybutyramido-d-fucose (1 part). From the results of methylation analysis, Smith-type periodate oxidation degradation studies, and the use of one- and two-dimensional (1)H and (13)C NMR spectroscopy, the O-polysaccharide antigen was found to be an unbranched polymer of a repeating pentasaccharide unit having the following structure: -->2)-Beta-d-Glcp-(1-->2)-Beta-d-Fucp3NBu-(1-->6)-alpha-d-GlcpNAc-(1-->4)-alpha-d-GalpNAc-(1-->3)-Beta-d-GlcpNAc-(1-->,where Bu is (R)-3-hydroxybutyramido.

Identification and differentiation of Taylorella equigenitalis and Taylorella asinigenitalis by lipopolysaccharide O-antigen serology using monoclonal antibodies.

Lipopolysaccharides (LPSs) from Taylorella equigenitalis, the causative agent of contagious equine metritis, and T. asinigenitalis were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide profiles of 11 T. equigenitalis strains were similar, but different from the profiles of 3 T. asinigenitalis strains, and the profiles of 2 T. asinigenitalis strains were similar to each other. The serological specificities of the LPSs from these 14 strains were examined by immunoblotting and enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) to the LPSs of the T. equigenitalis and T. asinigenitalis type strains and T. asinigenitalis strain 2329-98. A MAb to T. equigenitalis LPS O-polysaccharide (O-PS) (M2560) reacted with LPSs from all T. equigenitalis strains but did not react with LPSs from the 3 T. asinigenitalis strains or with 43 non-Taylorella bacteria. Three MAbs to the T. asinigenitalis type strain LPS O-PS or core epitopes (M2974, M2982, M3000) reacted with the homologous strain and T. asinigenitalis strain Bd 3751/05, but not with any of the other bacteria. Five MAbs to T. asinigenitalis 2329-98 LPS O-PS or core epitopes (M2904, M2907, M2910, M2923, M2929) reacted only with this strain. Proton nuclear magnetic resonance spectra of the O-PSs of the type strains of T. equigenitalis and T. asinigenitalis provided fingerprint identification and differentiation of these 2 organisms. The serological results were consistent with our previous finding that the O-antigen of the type strain of T. equigenitalis, being a linear polymer of disaccharide repeating [-->4)-alpha-L-GulpNAc3NAcA-(1-->4)-beta-D-ManpNAc3NAcA-(1-->] units, differs from that of the T. asinigenitalis O-antigen polymer that is composed of repeating [-->3)-beta-D-QuipNAc4NAc-(1-->3)-beta-D-GlcpNAmA-(1-->] units. Lipopolysaccharide O-PS could be a specific marker for identification and differentiation of T. equigenitalis and T. asinigenitalis, and provide the basis for the development of specific detection assays for T. equigenitalis.

Characterization of the antigenic O-polysaccharide produced by Escherichia coli serotype O:70.

The structure of the antigenic O-polysaccharide (O-PS) produced by Escherichia coli serotype O:70 was determined by analysis of the chromatographically purified O-PS polymer prepared by mild hydrolysis of its aqueous phenol-extracted smooth-type somatic lipopolysaccharide. The O-PS is composed of D-glucose, D-galactose, D-fucose, 2-acetamido-2-deoxy-D-galactose, and 3-acetamido-3-deoxy-D-quinovose in a ratio of 1:1:1:1:1. From the use of DOC-PAGE, methylation, Smith-type periodate oxidation, and (1)H and (13)C NMR spectroscopy, including 2D experiments, the O-PS was shown to be a polymer of a branched repeating pentasaccharide unit having the structure: [structure: see the text]

Characterization of the O-antigen in the lipopolysaccharide of Cronobacter (Enterobacter) malonaticus 3267.

Cronobacter malonaticus, a Gram-negative bacterium previously known as Enterobacter sakazakii, is an opportunistic pathogen known to cause serious infection in infants and neonates. To provide aid for the serological and chemical identification of clinical, environmental, or food isolates of this emerging pathogen, the characterization of the lipopolysaccharide (LPS) O-polysaccharide (O-PS) antigens of Cronobacter spp. is being undertaken. The structural analysis of the O-PS, obtained by hydrazinolysis of the lipopolysaccharide produced by Cronobacter malonaticus HPB 3267, was investigated by composition, methylation, and two-dimensional high-resolution nuclear magnetic resonance methods, and was found to be a polymer of a branched pentasaccharide unit. This unit is composed of D-glucose (D-Glc), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN) and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues (1: 1: 1: 1: 1), forms the repeating oligosaccharide in the O-PS antigen, and has the structure: [structure: see text].

Structure of the antigenic repeating pentasaccharide unit of the LPS O-polysaccharide of Cronobacter sakazakii implicated in the Tennessee outbreak.

Strains of the Gram-negative bacterium Cronobacter (Enterobacter) sakazakii have been identified as emerging opportunistic pathogens that can cause enterocolitis, bacteraemia, meningitis, and brain abscess, and have been particularly associated with meningitis in neonates where infant-milk formulae has been epidemiologically linked to the disease. A study of the lipopolysaccharides produced by clinical isolates using chemical, 2D 1H and 13C NMR, and MS methods revealed that the O-polysaccharide produced by C. sakazakii (3290), a clinical strain from the Tennessee outbreak, was a branched polymer of repeating pentasaccharide units composed of 2-acetamido-2-deoxy-D-galactose, 3-(N-acetyl-L-alanylamido)-3-deoxy-D-quinovose, D-glucuronic acid, and D-glucose present in the molar ratio 1:1:1:2 and had the structure:

The structure of the O-antigen in the endotoxin of the emerging food pathogen Cronobacter (Enterobacter) muytjensii strain 3270.

Strains of the Gram-negative bacterium Cronobacter (formerly known as Enterobacter) sakazakii have been identified as emerging opportunistic pathogens that can cause enterocolitis, bacteraemia, meningitis, and brain abscess, and they have been particularly associated with meningitis in neonates where infant milk formulae have been epidemiologically linked to the disease. A study of the lipopolysaccharides produced by clinical isolates using chemical, 2D 1H and 13C NMR, and MS methods revealed that the O-polysaccharide produced by Cronobacter muytjensii strain 3270, isolated from powdered infant formula from Denmark, was a linear unbranched polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-d-galactose (d-GalNAc), 2-acetamido-2-deoxy-d-glucose (d-GlcNAc), 3-acetamido-3-deoxy-d-quinovose (d-Qui3NAc), l-rhamnose (l-Rha), and d-glucuronic acid (d-GlcA) in equimolar ratio, and has the structure -->3)-alpha-D-GalpNAc-(1-->4)-alpha-D-Quip3NAc-(1-->3)-alpha-L-Rhap-(1-->6)-alpha-D-GlcpNAc-(1-->4)-beta-D-GlcpA-(1--> The specific structural characteristics of the O-polysaccharides of C. muytjensii may be of value in the identification and tracking of the bacterial pathogen.

The structure of the polysaccharide O-chain of the LPS from Acinetobacter baumannii strain ATCC 17961.

The gram-negative bacterium Acinetobacter baumannii strain ATCC17961 has been used by several laboratories in mouse models of respiratory A. baumannii infection, and a study of the role of its lipopolysaccharide in the pathogenicity is of interest. The structure of the O-deacylated polysaccharide O-chain component of its LPS has been determined by 2D NMR spectroscopy and mass spectrometry methods, and by the structural identification of oligosaccharides obtained by sequential application of the Smith degradation of the O-antigen. The O-chain was determined to be a polymer of a branched pentasaccharide repeating unit composed of 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-glucose, and D-galactose, and has the following structure: [carbohydrate sequence see in text].

The structure of the polysaccharide of the lipopolysaccharide produced by Taylorella equigenitalis type strain (ATCC 35865).

Taylorella equigenitalis is a Gram-negative bacterium that causes venereally transmitted contagious equine metritis (CEM), and its identification and differentiation from other bacteria and Taylorella species is an important requirement for the control of CEM infection. Based on the results of NMR and MS analysis, the antigenic O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) produced by the type strain T. equigenitalis (ATCC 35865) was found to be a linear polymer composed of a repeating disaccharide unit, containing partially amidated 2,3-diacetamido-2,3-dideoxy-alpha-L-guluronic and 2,3-diacetamido-2,3-dideoxy-beta-D-mannuronic acids, terminated with a 4-O-methylated non-reducing Gulp-NAc3NAcA residue, and has the structure [structure: see text]. The O-PS of the type strain T. equigenitalis LPS provides a specific antigenic marker for the discrimination of the pathogen from the related type strain of T. asinigenitalis sp. nov, a phenotypically indistinguishable non-pathogenic bacterium having a serologically and structurally unrelated LPS O-antigen. The analysis of a structurally unusual core oligosaccharide of the LPS is also reported.

Structure of the O-polysaccharide of the lipopolysaccharide produced by Taylorella asinigenitalis type strain (ATCC 700933).

Taylorella asinigenitalis sp. nov is a nonpathogenic gram-negative bacterium recently isolated from the genital tract of male donkeys. The bacterium is phenotypically indistinguishable from Taylorella equigenitalis, a pathogen that is the cause of contagious equine metritis, a highly communicable venereal disease of horses. The structural analysis of the lipopolysaccharide produced by T. asinigenitalis sp. nov (ATCC 700933) demonstrated that its O-polysaccharide (O-PS) component is a linear unbranched polymer of repeating disaccharide units composed of 1,3-linked pyranosyl residues of 2,4-diacetamido-2,4-dideoxy-beta-D-quinovose (bacillosamine) and 2-acetamidino-2-deoxy-beta-D-glucuronic acid, and has the structure [-->3)-beta-D-QuipNAc4NAc-(1-->3)-beta-D-GlcpNAmA-(1-->]n. The chemical structure and serological characteristics of the T. asinigenitalis O-PS are distinct from those of the O-PS of the T. equigenitalis type strain, thus providing a cell-surface target macromolecule that can be used to distinguish pathogenic from nonpathogenic Taylorella sp. clinical isolates.

The structure of the exocellular polysaccharide produced by Rhodococcus sp. RHA1.

Rhodococcus sp. RHA1 is a Gram-positive actinomycete capable of metabolizing a wide spectrum of organic compounds whose survival in chemically hostile environments is believed to be in part due to the production of an exocellular polysaccharide (EPS). In order to investigate the functional nature of the EPS, its structure was determined using a combinatory approach including hydrolysis, composition, and methylation, analysis methods, as well as 2D (1)H and (13)C NMR spectroscopy. The EPS was found to be a high-molecular-mass polymer of a repeating tetrasaccharide unit composed of D-glucuronic acid, D-glucose, D-galactose, L-fucose and O-acetyl (1:1:1:1:1), and has the structure:

Structural elucidation of the O-antigenic polysaccharide from enterohemorrhagic (EHEC) Escherichia coli O48:H21.

The structure of the antigenic O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) produced by the enterohemorrhagic strain of Escherichia coli O48:H21 (EHEC) has been elucidated. The O-PS obtained by mild acid hydrolysis of the LPS had [alpha]D +95 (water) and was composed of L-rhamnose (L-Rha), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN), and D-galacturonic acid (D-GalA) (1:1:1:1:1). From the results of methylation analysis, mass spectrometry, 2D NMR, and DOC-PAGE, the O-PS was shown to be a high molecular mass polymer of a repeating pentasaccharide unit having the structure: [structure: see text]. The D-Gal pA non-reducing end groups in the O-PS were partially O-acetylated (approximately 30%) at the O-2 and O-3 positions and the degree of acetylation was variable from batch to batch cell production.

Structural characterization of the antigenic capsular polysaccharide and lipopolysaccharide O-chain produced by Actinobacillus pleuropneumoniae serotype 15.

The specific capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 15 was determined to be a high-molecular-mass polymer having [alpha]D + 69 degrees (water) and composed of a linear backbone of phosphate diester linked disaccharide units of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc) and 2-acetamido-2-deoxy-D-galactose (D-GalNAc) residues (1:1). Thirty percent of the D-GalNAc residues were substituted at O-4 by beta-D-galactopyranose (beta-D-Galp) residues. Through the application of chemical and NMR methods, the capsule, which defines the serotype specificity of the bacterium, was found to have the structure [structure: see text]. The O-polysaccharide (O-PS) component of the A. pleuro pneumoniae serotype 15 lipopolysaccharide (LPS) was characterized as a linear unbranched polymer of repeating pentasaccharide units composed of D-glucose (2 parts) and D-galactose (3 parts), shown to have the structure [structure: see text]. The O-PS was chemically identical with the O-antigen previously identified in the LPSs produced by A. pleuro pneumoniae serotypes 3 and 8.