Structure of an O-specific polysaccharide of Proteus mirabilis 03, containing N-(2-hydroxyethyl)-D-alanine

O-Specific polysaccharide, obtained by mild acid degradation of the Proteus mirabilis 03 lipopolysaccharide, 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, the 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 elucidated by using 1H and 13C NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative), optical rotation and CD spectrum data and comparison with the synthetic sample. The repeating unit of P. mirabilis 03 O-specific polysaccharide is shown to have the following structure: (formula; see text)     

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:     

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-specific polysaccharide of the bacterium Proteus vulgaris O23

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Proteus vulgaris O23 (strain PrK 44/57) and found to contain 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-galacturonic acid. Based on 1H- and 13C-NMR spectroscopic studies, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [figure], where the degree of O-acetylation of the terminal GalA residue at position 4 is about 80%. A structural similarity of the O-specific polysaccharides of P. vulgaris O23 and P. mirabilis O23 is discussed.     

Structural and serological studies on the O-antigen of Proteus mirabilis O14, a new polysaccharide containing 2-[(R)-1-carboxyethylamino]ethyl phosphate.

An O-specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain D-galactose, 2-acetamido-2-deoxy-D-glalactose, phosphate, N-(2-hydroxyethyl)-D-alanine (D-AlaEtn), and O-acetyl groups. Studies of the initial and O-deacetylated polysaccharides using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, NOESY, H-detected 1H,13C heteronuclear multiple-quantum coherence, and heteronuclear multiple-bond correlation experiments, demonstrated the following structure of the repeating unit: [equation: see text] This is the second bacterial polysaccharide reported to contain alpha-D-Galp6PAlaEtn, whereas the first one was the O-antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska-Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem., 188, 645-651]. Anti-(P. mirabilis O14) serum cross-reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O-antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O-antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.     

The structure of O-specific polysaccharide of Proteus mirabilis 027 containing amino acids and phosphoethanolamine

The following structure of the repeating unit of the Proteus mirabilis O27 O-specific polysaccharide was established: (formula; see text) where (formula; see text) is N-glucopyranuronoyl-L-lysine, (formula; see text) is N-galactopyranuronoyl-L-alanine. The polysaccharide was parially solvolysed with anhydrous HF and the resulting dephosphorylated tri- and tetrasaccharide with N-acetylglucosamine at the reducing end were studied by means of 1H and 13C NMR spectroscopy and (for methylated derivative of trisaccharide) mass-spectrometry. Smith degradation of the polysaccharide afforded linear polymer, and its structure was investigated by 13C NMR spectroscopy. The position of the ethanolamine phosphate group was determined by means of the analysis of the phosphorylation effects in the 13C NMR spectra of the linear and branched polysaccharides.     

Isolation using triflic acid solvolysis and identification of N(epsilon)-[(R)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine as a component of the O-specific polysaccharide of Proteus mirabilis O13

An amino acid was released from the O-specific polysaccharide of Proteus mirabilis O13 by acid hydrolysis and identified as N(epsilon)-[(R)-1-carboxyethyl]-L-lysine by comparison with the authentic sample. An amide of this amino acid with D-galacturonic acid was isolated from the polysaccharide by solvolysis with anhydrous trifluoromethanesulfonic (triflic) acid and characterised by 1H and 13C NMR spectroscopy. These and published data enabled determination of the full structure of the repeating unit of the polysaccharide.     

Structure of the O-specific polysaccharide of Proteus mirabilis D52 and typing of this strain to Proteus serogroup O33.

The acidic O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H-NMR and 13C-NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain D-ribitol 5-phosphate (D-Rib-ol-5-P) and ethanolamine phosphate (Etn-P) and has the following structure: D-Rib-ol-5-P (3) approximately 75% EtnP(6)-->2)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-GlcpNAc-(1-->). This structure is identical with that of the O-polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O-antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross-reactivity of P. mirabilis D52 O-antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O-polysaccharides. The role of charged groups, Rib-ol-5-P and Etn-P in the immunospecificity is discussed.     

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.     

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.     

O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770

The O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770 was obtained by mild acid degradation of the corresponding lipopolysaccharide and found to contain D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine residues in equimolar ratio. Based on methylation analysis, periodate oxidation, and 13C-NMR spectroscopy data of native and modified polysaccharides, the following structure of the trisaccharide repeating unit of the O-specific polysaccharide was established: [structure: see text]     

Full structure of the O-specific polysaccharide of Proteus mirabilis O24 containing 3,4-O-[(S)-1-carboxyethylidene]-D-galactose

The structure of a neutral polysaccharide isolated by degradation with dilute acetic acid of the lipopolysaccharide (LPS) of P. mirabilis O24 has been determined recently [E. Literacka et al., FEBS Lett., 456 (1999) 227-231]. Further studies of this LPS using alkaline degradation and hydrolysis at pH 4.5 showed that the polysaccharide chain includes an acetal-linked pyruvic acid residue, which is removed completely during delipidation with acetic acid. A revision using 1H and 13C NMR spectroscopy and methylation analysis resulted in determination of the following full structure of the repeating unit of the O-specific polysaccharide: carbohydrate sequence [see text] where D-Gal3,4(S-Pyr) is 3,4-O-[(S)-1-carboxyethylidene]-D-galactose.     

Structure of an acidic O-specific polysaccharide of the bacterium Providencia alcalifaciens O7

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O7 and purified by gel chromatography followed by anion-exchange chromatography. On the basis of full acid hydrolysis, methylation, carboxyl reduction, selective cleavage with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional 1H,1H homonuclear and H-detected 1H,13C heteronuclear correlation spectroscopy and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: [figure], where Rhap2Ac is 2-O-acetylrhamnopyranose.     

Structural and serological studies of the related O-specific polysaccharides of Proteus vulgaris O21 and Proteus mirabilis O48 having oligosaccharide-phosphate repeating units.

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides (LPSs) of Proteus mirabilis O48 and Proteus vulgaris O21 were found to have tetrasaccharide and pentasaccharide repeating units, respectively, interlinked by a glycosidic phosphate. Polysaccharides and an oligosaccharide were derived from the LPSs by various degradation procedures and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,13C and 1H,31P HMQC experiments. The following related structures of the repeating units of the O-antigens were established (top: Proteus mirabilis O48; bottom: Proteus vulgaris O21) The O-specific polysaccharide of P. vulgaris O21 has the same structure as that of Hafnia allvei 744 and PCM 1194 [Petersson C., Jachymek, W., Klonowska, A., Lugowski, C., Niedziela, T. & Kenne, L. (1997) Eur. J. Biochem., 245, 668-675], except that the GlcN residue carries the N-acetyl rather than the N-[(R)-3-hydroxybutyryl] group. Serological investigations confirmed the close relatedness of the Proteus and Hafnia O-antigens studied.     

The structure of the O-specific polysaccharide chain of Proteus penneri strain 16 lipopolysaccharide

O-Specific polysaccharide was obtained by mild acid degradation of Proteus penneri strain 16 lipopolysaccharide and found to contain D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]- D-galactose in the ratio of 2:1:1:1 as well as a small proportion of O-acetyl groups. On the basis of one-dimensional 1H-NMR13C-NMR and NOE spectroscopy, two-dimensional homonuclear-shift-correlated spectroscopy with one-step and two-step relayed coherence transfer and heteronuclear 1H/13C NMR shift-correlated spectroscopy, it was concluded that the O-specific polysaccharide of P. penneri strain 16 has the following structure: (formula; see text) This structure was confirmed by methylation analysis and structural analysis of a linear tetrasaccharide fragment prepared by cleavage of the polysaccharide with anhydrous hydrogen fluoride followed by conversion of the alpha-tetrosyl fluoride obtained in to the corresponding free oligosaccharide and alditol. O-Acetyl groups were tentatively located at position 3 of the glucuronic acid residue and at position 4 of the 6-substituted glucose residue, the degree of acetylation being less than 20% of the total. Cross-reactions of P. penneri strain 16 anti-(O-specific polysaccharide) antiserum with lipopolysaccharides from several other Proteus strains and the role of 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-galactose in the serological specificity of P. penneri strain 16 are discussed.     

Antigenic polysaccharides of Shigella bacteria. Structure of the polysaccharide chain of the lipopolysaccharide from Shigella boydii, type 11]

On mild acid degradation of the Shigella boydii, type 11 lipopolysaccharide, the corresponding O-specific polysaccharide composed of D-glucuronic acid, 2-acetylamino-2-deoxy-D-glucose, D-ribose and L-rhamnose residues in the ratio 1:1:1:3 was obtained. Methylation, partial acid hydrolysis and 13C-NMR spectral data for the polysaccharide led to the structure of the oligosaccharide repeating unit as a branched hexasaccharide: [formula: see text]. Numerous O-acetyl groups attached non-stoichiometrically to the residues of D-glucuronic acid, L-rhamnose and 2-acetylamino-2-deoxy-D-glucose were located with the use of 13C-NMR spectroscopy.     

Antigenic bacterial polysaccharides. 13. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from Pseudomonas cepacia strain IMV 4137

On mild acid degradation of a lipopolysaccharide from Pseudomonas cepacia strain IMV 4137, a serologically active O-specific polysaccharide was obtained and shown to contain L-rhamnose and D-galactose. According to 1H- and 13C-NMR data as well as methylation analysis, the polysaccharide is made up of disaccharide repeating units of the following structure:----2)-alpha-L-Rhap-(1----4)-alpha-D-Galp-(1----.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Antigenic polysaccharides of bacteria. 25. Structure of the O-specific polysaccharide chain of Pseudomonas cepacia 673/2 lipopolysaccharide containing L-glycero-D-manno-heptose

O-Specific polysaccharide, consisting of D-rhamnose and L-glycero-D-manno-heptose (LD-Hep) in a 2 : 1 ratio, was obtained on the mild acid degradation of the Pseudomonas cepacia IMV 673/2 lipopolysaccharide; monosaccharide LD-Hep has not previously been found in O-specific chains of lipopolysaccharides. On the basis of methylation and 13C-NMR data, it was concluded that the polysaccharide is composed of trisaccharide repeating units having the following structure: ----3)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1----2)-alpha-LD-Hep-(1----     

Structure of the O-polysaccharide of Proteus mirabilis OC (CCUG 10702) from a new proposed Proteus serogroup O75

A neutral O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis OC (CCUG 10702) and studied by sugar and methylation analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text]. Based on the unique structure of the O-polysaccharide and serological data, we propose classifying P. mirabilis OC (CCUG 10702) into a new separate Proteus serogroup O75. A weak cross-reaction of O-antiserum against P. mirabilis OC with the lipopolysaccharide of P. mirabilis O49 was accounted for by a similarity in the O-polysaccharide structures.