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 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.     

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.     

The structure of the core part of Proteus vulgaris OX2 lipopolysaccharide

The identity of a novel structural component, an open-chain acetalic linkage, in the core part of the lipopolysaccharide (LPS) from Proteus vulgaris serotype OX2 has been determined by extensive NMR spectroscopic analysis of fragments isolated after mild acid hydrolysis of the intact LPS. The open-chain N-acetylgalactosamine fragment is substituted in the 4-position by non-stoichiometric amounts of a beta-galactopyranose residue and the overall structure of the core is as follows: [formula: see text] All sugars except the N-acetylgalactosamine are in the pyranose form, alpha-Hep refers to L-glycero-alpha-D-manno-heptopyranose and alpha-DDHep to D-glycero-alpha-D-manno-heptopyranose. Bold italics indicate non-stoichiometric substituents.     

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-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 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 and serological studies of the O-polysaccharide of Proteus penneri 75 Epitopes and subgroups of Proteus serogroup O73

The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 75 consists of tetrasaccharide-ribitol phosphate repeating units and resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was elucidated by chemical methods and 1H and 13C NMR spectroscopy: [structure in text] where Rib-ol is ribitol. Serological studies with polyclonal antisera showed that the same structure of the O-polysaccharide occurred in two strains: P. penneri 75 and 128. A similar structure has been established earlier for the O-polysaccharide of P. penneri 103 [Drzewiecka, D., et al., Carbohydr. Res. 337 (2002) 1535-1540]. On the basis of complex serological investigations with use of two polyclonal P. penneri 75 and 103 O-antisera, five strains could be classified into Proteus O73 serogroup: P. penneri 48, 75, 90, 103 and 128, two of which (P. penneri 75 and 128) should be subdivided into subgroup 73a, 73b and three others (P. penneri 48, 90 and 103) into subgroup 73a, 73c. Epitopes responsible for the cross-reactivity of P. penneri O73 strains and a related strain of P. mirabilis O20 were tentatively defined.     

The structure of the core part of Proteus penneri strain 16 lipopolysaccharide

The structure of the carbohydrate backbone of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus penneri strain 16 was determined using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of the LPS, and of the products of alkaline deacylation of the LPS: formula [see text]. Incomplete substitution is indicated by bold italics. All sugars are in the pyranose form, alpha-Hep is the residue of L-glycero-alpha-D-manno-Hep, alpha-DD-Hep is the residue of D-glycero-alpha-D-manno-Hep, Bu is the (R)-3-hydroxybutyryl residue.     

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.     

The structure of the carbohydrate backbone of the rough type lipopolysaccharides from Proteus penneri strains 12, 13, 37 and 44

The following structure of the lipid A-core backbone of the rough type lipopolysaccharides (LPS) from Proteus penneri strains 12, 13, 37, and 44 was determined using NMR and mass spectroscopy and chemical analysis of the oligosaccharides obtained by mild-acid hydrolysis, alkaline O,N-deacylation, O-deacylation with hydrazine, and deamination of the LPSs:where K=H, R=PEtN, R(1)=alpha-Hep-(1-->2)-alpha-DDHep, and R(2)=alpha-GalN (strains 12 and 13) or beta-GlcNAc-(1-->4)-alpha-GlcN (strains 37 and 44). LPS from each strain contained several structural variants. LPS from strain 12 contained a variant with R(1)=alpha-DDHep, whereas LPS from strains 13, 37, and 44 contained structures with K=amide of beta-GalA with putrescine or spermidine. The phosphate group at O-1 of the alpha-GlcN residue in the lipid part was partially substituted with Ara4N.     

The structure of the carbohydrate backbone of the core–lipid A region of the lipopolysaccharide from Proteus penneri strain 40: new Proteus strains containing open-chain acetal-linked N-acetylgalactosamine in the core part of the LPS

Analysis of the core part of the LPS from several strains of Proteus revealed that P. penneri strains 2, 11, 19, 107, and P. vulgaris serotypes O4 and O8 have the same structure with a new type of linkage between monosaccharides–an open-chain acetal — that was previously determined for P. vulgaris OX2 and P. penneri 17. The LPS from P. penneri strain 40 contains the same structure substituted with one additional monosaccharide:

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where (1S)-GalaNAc¹ is a residue of N-acetyl- -galactosamine in the open-chain form. It is connected as a cyclic acetal to positions 4 and 6 of the galactosamine residue having a free amino group. All other sugars are in the pyranose form.     

Investigation of hydrophobicity of Proteus vulgaris strains and ability of Proteus vulgaris and Proteus penneri strains to penetrate bladder membrane HCV T-29 cells

Proteus bacilli play a particularly important role in urinary tract infections (UTI). Fimbriae and adherence ability and hemolysins production (HpmA, HlyA) are one of the factors of pathogenicity of these bacteria. In this paper we describe the invasion of HCV T-29 transitional bladder urothelial cells carcinoma strains of P. penneri, as well as P. vulgaris strains belonging to different serogroups. The cytotoxic effect was observed at 8 hour of incubation of the tested cells with P. vulgaris O21 and the same effect (complete lysis) at 6 hours by P. vulgaris O4 (this strain manifests maximal activity in the production of HlyA hemolysin). P. penneri strains, produce different types of fimbriae, expressed similar bacterial invasiveness. The hydrophobic properties of 25 P. vulgaris strains were also tested and only 3 strains occur to have hydrophobic cell surface.     

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.     

Structural and serological studies of the O-antigen of Proteus mirabilis O-9

The following structure of the O-polysaccharide (O-antigen) of the lipopolysaccharide of Proteus mirabilis O-9 was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with chemical methods: [chemical structure: see text] where the degree of O-acetylation is approximately 70%. Immunochemical studies using rabbit polyclonal anti-Proteus mirabilis O-9 serum showed the importance of the O-acetyl groups in manifesting the serological specificity of the O-9 antigen. Anti-P. mirabilis O-9 cross-reacted with the lipopolysaccharides (LPS) of P. vulgaris O-25 and Proteus penneri 14, which could be accounted for by a structural similarity of their O-polysaccharides.     

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 a glycerol teichoic acid-like O-specific polysaccharide of Proteus vulgaris O12.

A phosphorylated O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of Proteus vulgaris O12 lipopolysaccharide and studied by sugar and methylation analyses, 1H-, 13C- and 31P-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H, 13C and 1H, 31P heteronuclear multiple-quantum coherence experiments. It was found that the polysaccharide consists of pentasaccharide repeating units connected via a glycerol phosphate group, and has the following structure: where FucNAc is 2-acetamido-2,6-dideoxygalactose and the degree of O-acetylation at position 4 of GalNAc is approximately 25%. Immunochemical studies with P. vulgaris O12 O-antiserum suggested that the lipopolysaccharide studied shares common epitopes with the lipopolysaccharide core of P. vulgaris O8 and with the O-antigens of P. penneri strains 8 and 63.     

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 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.     

Identification and typing of Proteus penneri and Proteus vulgaris biogroups 2 and 3, from clinical sources, by computerized analysis of electrophoretic protein patterns

Seventy-six strains of the Proteus vulgaris complex ( Pr. penneri and Pr. vulgaris biogroups 2 and 3) were characterized by one-dimensional SDS-PAGE of cellular proteins. The protein patterns were highly reproducible. The strains came from various countries and were mainly of human origin: urine (28), respiratory tract (13), wounds (8), faeces (7), blood (3), miscellaneous sources (6) and unknown sources (11). The patterns of these strains, together with those of the type strains of seven Morganella, Proteus and Providencia species were subjected to two numerical analyses. In the first, in which the principal protein bands (in the 35.0–42.0 kDa range) were excluded, the strains of the Pr. vulgaris complex formed four clusters at the 83% similarity level. These corresponded to Pr. penneri, Pr. vulgaris biogroup 2, and two clusters (3a and 3b) represented biogroup 3. Each of these clusters was distinct from the Morganella, Proteus and Providencia reference strains. In the second analysis, which included all the protein bands, the 41 Pr. penneri strains showed little heterogeneity but 17 subphenons could be recognized among the 35 strains of Pr. vulgaris biogroups 2 and 3. These results support the division of biogroup 3 strains into at least two separate taxa. Other results indicate that biogroup 3 is heterogeneous and may contain further genomic groups. The method also provides a basis for typing clinical strains of Pr. vulgaris biogroups 2 and 3.