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 Proteus penneri 103 containing ribitol and 2-aminoethanol phosphates

The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 103 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,(13)C HMQC, 1H, 31P HMQC, and HMBC experiments. It was found that the polysaccharide is built up of oligosaccharide-ribitol phosphate repeating units and thus resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was established:where Etn and Rib-ol are ethanolamine and ribitol, respectively. This structure is unique among the known structures of Proteus O-antigens and, therefore, we propose classification of the strain studied into a new Proteus serogroup, O73. The molecular basis for cross-reactivity between O-antiserum against P. penneri 103 and O-antigens of P. mirabilis O33 and D52 is discussed.     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus penneri 60 classified into a new Proteus serogroup O70

An alkali-treated lipopolysaccharide of Proteus penneri strain 60 was studied by chemical analyses and 1H, 13C and 31P NMR spectroscopy, and the following structure of the linear pentasaccharide-phosphate repeating unit of the O-polysaccharide was established: 6)-alpha-D-Galp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4NAc-(1-->6)-alpha-D-Glcp-1-P-(O--> Rabbit polyclonal O-antiserum against P. penneri 60 reacted with both core and O-polysaccharide moieties of the homologous LPS. Based on the unique O-polysaccharide structure and serological data, we propose to classify P. penneri 60 into a new, separate Proteus serogroup O70. A weak cross-reactivity of P. penneri 60 O-antiserum with the lipopolysaccharide of Proteus vulgaris O8, O15 and O19 was observed and discussed in view of the chemical structures of the O-polysaccharides.     

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 a new ribitol teichoic acid-like O-polysaccharide of a serologically separate Proteus vulgaris strain, TG 276-1, classified into a new Proteus serogroup O53

An unusual ribitol teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide from a previously non-classified Proteus vulgaris strain TG 276-1. Structural studies using chemical analyses and 2D (1)H and (13)C NMR spectroscopy showed that the polysaccharide is a zwitterionic polymer with a repeating unit containing 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N) and two D-ribitol phosphate (D-Rib-ol-5-P) residues and having the following structure:[formula: see text] where the non-glycosylated ribitol residue is randomly mono-O-acetylated. Based on the unique O-polysaccharide structure and the finding that the strain studied is serologically separate among Proteus bacteria, we propose to classify P. vulgaris strain TG 276-1 into a new Proteus serogroup, O53.     

Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72.

O-specific polysaccharides (O-antigens) of the lipopolysaccharides (LPS) of Proteus penneri strains 1 and 4 were studied using sugar analysis, (1)H and (13)C NMR spectroscopy, including 2D COSY, H-detected (1)H,(13)C HMQC, and rotating-frame NOE spectroscopy (ROESY). The following structures of the tetrasaccharide (strain 1) and pentasaccharide (strain 4) repeating units of the polysaccharides were established: [reaction: see text]. In the polysaccharide of P. penneri strain 4, glycosylation with the lateral Glc residue (75%) and O-acetylation of the lateral GalNAc residue (55%) are nonstoichiometric. This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined. The structural similarity of the O-specific polysaccharides was consistent with the close serological relatedness of the LPS, which was demonstrated by immunochemical studies with O-antisera against P. penneri 1 and 4. Based on these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two subgroups, O72a and O72a,b, respectively. Serological cross-reactivity of P. penneri 1 O-antiserum with the LPS of P. penneri 40 and 41 was substantiated by the presence of an epitope(s) on the LPS core region shared by all P. penneri strains studied.     

Structure of the O-polysaccharide of Proteus penneri 28 and Proteus vulgaris O31 and classification of P. penneri 26 and 28 in Proteus serogroup O31

The lipopolysaccharides (LPS) of Proteus penneri 28 and Proteus vulgaris O31 (PrK 55/57) were degraded with dilute acetic acid and structurally identical high-molecular-mass O-polysaccharides were isolated by gel-permeation chromatography. Sugar analysis and nuclear magnetic resonance (NMR) spectroscopic studies showed that both polysaccharides contain D-GlcNAc, 2-acetamido-2,6-dideoxy-L-glucose (L-2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine)) and 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (N-acetylisomuramic acid) and have the following structure: [carbohydrate structure: see text] where (S)-1-carboxyethyl [a residue of (S)-lactic acid] (S-Lac) is an ether-linked residue of (S)-lactic acid. The O-polysaccharide studied is structurally similar to that of P. penneri 26, which differs only in the absence of S-Lac from the GlcNAc residue. Based on the O-polysaccharide structures and serological data of the LPS, it was suggested classifying these strains in one Proteus serogroup, O31, as two subgroups: O(31a), 31b for P. penneri 28 and P. vulgaris PrK 55/57 and O31a for P. penneri 26. A serological relatedness of the LPS of Proteus O(31a), 31b and P. penneri 62 was revealed and substantiated by sharing epitope O31b, which is associated with N-acetylisomuramic acid. It was suggested that a cross-reactivity of P. penneri 28 O-antiserum with the LPS of several other P. penneri strains is due to a common epitope(s) on the LPS core.     

Structure and serological properties of the O-antigen of two clinical Proteus mirabilis strains classified into a new Proteus O77 serogroup

Two Proteus mirabilis strains, 3 B-m and 3 B-k, were isolated from urine and faeces of a hospitalized patient from Lodz, Poland. It was suggested that one strain originated from the other, and the presence of the bacilli in the patient's urinary tract was most probably a consequence of autoinfection. The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of P. mirabilis 3 B-m and studied by sugar analysis and nuclear magnetic resonance spectroscopy, including two-dimensional rotating frame Overhause effect spectroscopy (ROESY) and 1H,13C heteronuclear single quantum coherence (HSQC) experiments. The following structure of the linear trisaccharide-repeating unit of the O-polysaccharide was established:-->2)-beta-D-Glcp-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where 6dTal2Ac stands for 2-O-acetyl-6-deoxy-L-talose. It resembles the structure of the O-polysaccharide of Proteus penneri O66, which includes additional lateral residues of 2,3-diacetamido-2,3,6-trideoxy-L-mannose. The lipopolysaccharides from two P. mirabilis strains studied were serologically identical to each other but not to that from any of the existing 76 Proteus O-serogroups. Therefore, the strains were classified into a new O77 serogroup specially created in the genus Proteus. Serological studies using Western blot and enzyme-linked immunosorbent assay with intact and adsorbed O-antisera showed that the P. mirabilis O77 antigen is related to Proteus vulgaris O2 and P. penneri O68 antigens, and a putative disaccharide epitope responsible for the cross-reactivity was revealed.     

Structure of the O-polysaccharide of a serologically separate strain of Proteus mirabilis, TG 332, from a new proposed Proteus serogroup O50

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis TG 332 strain. The following structure of the O-polysaccharide was determined by chemical methods along with NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments: [see equation in text]. The O-polysaccharide studied has a unique structure among Proteus O-antigens. Accordingly, P. mirabilis TG 332 is serologically separate, and we propose to classify this strain into a new Proteus serogroup, O50. The nature of minor epitopes that provide a cross-reactivity of P. mirabilis TG 332 O-antiserum with the LPS of P. mirabilis O30 and Proteus penneri 34 (O60) is discussed.     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus mirabilis 2002

The structure of the O-polysaccharide of the lipopolysaccharide of Proteus mirabilis 2002 was elucidated by chemical methods and 1H and 13C NMR spectroscopy. It was found that the polysaccharide consists of branched pentasaccharide repeating units having the following structure: [structure in text]. The O-polysaccharide of P. mirabilis 2002 has a common tetrasaccharide fragment with that of P. mirabilis 52/57 from serogroup O29, and the lipopolysaccharides of the two strains are serologically related. Therefore, based on the structural and serological data, we propose to classify P. mirabilis 2002 into the Proteus O29 serogroup as a subgroup O29a,29b.     

Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3.

The O-chain polysaccharide of the lipopolysaccharide (LPS) of a previously nonclassified strain of Proteus mirabilis termed G1 was studied by sugar analysis and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, rotating-frame NOE (ROESY), H-detected 1H,13C HMQC, and heteronuclear multiple-bond correlation (HMBC) experiments. The following structure of the polysaccharide was established: [carbohydrate structure: see text] where D-GalA6(L-Lys) stands for N(alpha)-(D-galacturonoyl)-L-lysine. The structure of the O-polysaccharide of P. mirabilis G1 is similar, but not identical, to that of P. mirabilis S1959 and OXK belonging to serogroup O3. Immunochemical studies with P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera revealed close LPS-based serological relatedness of P. mirabilis G1 and S1959, and therefore it was suggested to classify P. mirabilis G1 in serogroup O3 as a subgroup. P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera also cross-reacted with LPS of P. mirabilis strains from two other serogroups containing D-GalA6(L-Lys) in the O-polysaccharide or in the core region.     

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 Proteus penneri 71 and classification of cross-reactive P. penneri strains to a new proposed serogroup O64.

A neutral O-specific polysaccharide (O-antigen) was isolated from the lipopolysaccharide (LPS) of the bacterium Proteus penneri 71. On the basis of sugar analysis and 1H- and 13C-NMR spectroscopic studies, including two-dimensional COSY, 13C,1H heteronuclear COSY and ROESY, the following structure of the trisaccharide repeating unit of the polysaccharide was established: -->3)-beta-D-GlcpNAc-(1-->4)-beta-D-GlcpNAc-(1-->3)-alpha-D-Galp-(1-- > The polysaccharide has the same carbohydrate backbone as the O-specific polysaccharide of P. penneri 19 and both are similar to that of P. penneri 62 studied by us previously. A cross-reactivity of anti-P. penneri 71, 19 and 62 O-antisera with 11 P. penneri strains was revealed and substantiated at the level of the O-antigen structures. These strains could be divided into three subgroups within a new proposed Proteus O64 serogroup containing P. penneri strains only.     

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.     

Structure of the N-acetyl-L-rhamnosamine-containing O-polysaccharide of Proteus vulgaris TG 155 from a new Proteus serogroup, O55

The O-polysaccharide of the lipopolysaccharide (LPS) of Proteus vulgaris TG 155 was found to contain 2-acetamido-2,6-dideoxy-L-mannose (N-acetyl-L-rhamnosamine, L-RhaNAc), a monosaccharide that occurs rarely in Nature. The following structure of the O-polysaccharide was established by NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H,13C HSQC experiments, along with chemical methods: [carbohydrate structure in text] Rabbit polyclonal O-antiserum against P. vulgaris TG 155 reacted with both core and O-polysaccharide moieties of the homologous LPS but showed no cross-reactivity with other LPS from the complete set of serologically different Proteus strains. Based on the unique O-polysaccharide structure and the serological data, we propose classifying P. vulgaris TG 155 into a new, separate Proteus O-serogroup, O55.     

Structure of the glycerol phosphate-containing O-polysaccharides and serological studies of the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 classified into a new Proteus serogroup, O54

O-Polysaccharides were obtained from the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 and studied by chemical analyses and one- and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy, including rotating-frame nuclear Overhauser effect spectroscopy, H-detected (1)H,(13)C heteronuclear single-quantum spectroscopy and (1)H,(31)P heteronuclear multiple-quantum spectroscopy experiments. The Proteus mirabilis OE polysaccharide was found to have a trisaccharide repeating unit with a lateral glycerol phosphate group. The Proteus vulgaris TG 103 produces a similar O-polysaccharide, which differs in incomplete substitution with glycerol phosphate (c. 50% of the stoichiometric amount) and the presence of an O-acetyl group at position 6 of the 2-acetamido-2-deoxygalactose (GalNAc) residue. These structures are unique among the known bacterial polysaccharide structures. Based on the structural and serological data of the lipopolysaccharides, it is proposed to classify both strains studied into a new Proteus serogroup, O54, as two subgroups, O54a,54b and O54a,54c. The serological relatedness of the Proteus O54 and some other Proteus lipopolysaccharides is discussed.     

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.     

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.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups

An acidic branched O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Proteus genomospecies 4 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected 1H, 13C HSQC experiments. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established, which is unique among Proteus polysaccharide structures: [structure: see text] where Qui3NAc stands for 3-acetamido-3,6-dideoxyglucose. Based on the O-polysaccharide structure and serological data, we propose classifying Proteus genomospecies 4 into a new, separate Proteus serogroup, O56. A weak cross-reactivity of Proteus genomospecies 4 antiserum with LPS of Providencia stuartii O18 and Proteus vulgaris OX2 was observed and is discussed in view of a similarity of the O-polysaccharide structures. Structural and serological investigations showed that Proteus genomospecies 5 and 6 should be classified into the existing Proteus serogroups O8 and O69, respectively.