Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.     

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.     

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 core part of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21

The core-lipid A region of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21 was studied using NMR spectroscopy, ESI MS, and chemical analysis after alkaline deacylation, deamination, and mild-acid hydrolysis of the lipopolysaccharides. The following general structure of the major core oligosaccharides is proposed: [abstract: see text] where all sugars are in the pyranose form and have the D configuration unless otherwise stated, Hep and DDHep=L-glycero- and D-glycero-D-manno-heptose, respectively, K=H, and Q=H in strain 8 or alpha-Glc in strains 7, 14, 15, and 21. In addition, several minor structural variants are present, including those lacking Ara4N in strains 7 and 15 and having the alpha-GlcN residue N-acylated to a various degree with glycine in strains 7, 8, 14, and 21. In strain 14, there are also core oligosaccharides with K=amide of beta-D-GalpA with putrescine, spermidine, or 4-azaheptane-1,7-diamine; remarkably, these structural variants lack either the PEtN group or the alpha-Hep-(1-->2)-alpha-DDHep disaccharide fragment at alpha-D-GalpA. While structural features of the inner core part are shared by Proteus strains studied earlier, the outermost Q-(1-->4)-alpha-GalNAc-(1-->2)-alpha-DDHep-(1-->6)-alpha-GlcN oligosaccharide unit has not been hitherto reported.     

Structure of the O-polysaccharide of Proteus serogroup O34 containing 2-acetamido-2-deoxy-alpha-D-galactosyl phosphate

On mild acid degradation of the lipopolysaccharide of Proteus vulgaris O34, strain CCUG 4669, the O-polysaccharide was cleaved at a glycosyl-phosphate linkage that is present in the main chain. The resultant phosphorylated oligosaccharides and an alkali-treated lipopolysaccharide were studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, and the following structure of the branched tetrasaccharide phosphate repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text]The O-polysaccharide of Proteus mirabilis strain TG 276 was found to have the same structure and, based on the structural and serological data, this strain was proposed to be classified into the same Proteus serogroup O34.     

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-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 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.     

Serotyping of clinical isolates belonging to Proteus mirabilis serogroup O36 and structural elucidation of the O36-antigen polysaccharide

The O-specific polysaccharide (OPS) isolated from the lipopolysaccharide of Proteus mirabilis O36 was found to have a pentasaccharide repeating unit of the following structure: -->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAc6Ac-(1-->4)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. The structure is unique among Proteus OPS, which is in agreement with the classification of this strain into a separate Proteus O-serogroup. Remarkably, the P. mirabilis O36-polysaccharide has the same structure as the OPS of Escherichia coli O153, except that the latter is devoid of O-acetyl groups. The cross-reaction of anti-O36 antibodies with the O-part of E. coli O153 lipopolysaccharide is observed. In the present study, two steps of serotyping Proteus strains are proposed: screening of dry mass with enzyme-linked immunosorbent assay and immunoblot with the crude lipopolysaccharides. This method allowed serotyping of 99 P. mirabilis strains infecting the human urinary tract. Three strains were classified into serogroup O36. The migration pattern of these lipopolysaccharides fraction with long O-specific PSs was similar to the standard laboratory P. mirabilis O36 (Prk 62/57) lipopolysaccharide. The relatively low number of clinical strains belonging to serogroup O36 did not correspond to the presence of anti-P. mirabilis O36 antibodies in the blood donors' sera. Twenty-five percent of tested sera contained a statistically significant elevated level of antibodies reacting with thermostable surface antigens of P. mirabilis O36. The presence and amount of antibodies correlated with Thr399Ile TLR4 polymorphism types (P=0.044).     

Structure of the O-polysaccharide of Proteus vulgaris O44: a new O-antigen that contains an amide of D-glucuronic acid with L-alanine

The O-polysaccharide of Proteus vulgaris O44, strain PrK 67/57 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H, 13C HMQC, HMQC-TOCSY and HMBC experiments. The polysaccharide was found to contain an amide of D-glucuronic acid with L-alanine [D-GlcA6(L-Ala)], and the following structure of the linear pentasaccharide repeating unit was established: [structure: see text]. The structural data of the O-polysaccharide and the results of serological studies with P. vulgaris O44 O-antiserum showed that the strain studied is unique among Proteus bacteria, which is in agreement with its classification in a separate Proteus serogroup, O44.     

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.     

The core region of Proteus mirabilis R110/1959 lipopolysaccharide

The complete core structure present in the lipopolysaccharide of the R mutant R110/1959 from Proteus mirabilis (Proteus type II core) was investigated using methylation analysis and a number of degradation methods such as Smith degradation and beta-elimination. These studies combined with earlier work on a Rc-type mutant of P. mirabilis O28 (R4/O28) which shares the same inner core region, allowed formulation of the complete core structure of the Proteus type II core as shown in Scheme 1. (formula; see text) A characteristic feature of the Proteus core of type II is the presence of two units of D-galacturonic acid (DGalA); one in terminal, the other one in a chain-linked position. In addition, the presence of the two isomers of glycero-D-manno-heptose (LDHep and DDHep) and the lack of galactose are conspicuous. DDHep occupies a terminal position in the external core region, whereas the three units of LDHep in addition to dOclA form, as in other enterobacterial core types, the internal core region. The taxonomic significance of the presence of DGalA in the Proteus type II core, but also in all R cores of other Proteeae investigated so far, will be discussed.     

Structure of the O-specific polysaccharide of Proteus vulgaris O37 and close serological relatedness of the lipopolysaccharides of P. vulgaris O37 and P. vulgaris O46

The O-specific polysaccharide (O-antigen) of the lipopolysaccharide (LPS) of Proteus vulgaris O37 was studied by (1)H and (13)C nuclear magnetic resonance spectroscopy before and after O-deacetylation and found to be structurally similar to that of P. vulgaris O46 studied earlier. The two polysaccharides have the same carbohydrate backbone and differ in the position and number of the O-acetyl groups only. Studies with O-antisera against the two strains using passive hemolysis test, enzyme immunosorbent assay, and Western blot revealed close serological relatedness of the LPSs of P. vulgaris O37 and O46. The O-acetyl groups were found to be of little importance for manifesting the O-specificity but to interfere with binding of anti-P. vulgaris O37 serum to P. vulgaris O46 antigen. Based on the data obtained, it was proposed to combine the strains studied in one Proteus serogroup O37 as subgroups O37a,37b and O37a,37c. A cross-reactivity of O-antisera against P. vulgaris O37 and O46 was observed with LPSs of three more Proteus strains, which could be substantiated by the presence of a common disaccharide fragment in the O-antigens.     

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 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 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 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.     

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 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 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.