Structure of a peptidoglycan-related polysaccharide from Providencia alcalifaciens O45.

A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional ROESY and H-detected (1)H,(13)C HSQC experiments. The polysaccharide contains N-acetylglucosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure: →4)-ß-D-GlcpNAc-(1→4)-ß-D-GlcpNAc3(Rlac-L-Ala)-(1→. The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.

Structure of the O-polysaccharide of Providencia alcalifaciens O25 containing an amide of D-galacturonic acid with N(ε)-[(R)-1-carboxyethyl]-L-lysine.

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O25 followed by gel-permeation and anion-exchange chromatography. The O-polysaccharide was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional correlation (1)H,(13)C HMBC, and (1)H,(1)H ROESY experiments both in D(2)O and, to detect correlations for NH protons, in a 9 : 1 H(2)O/D(2)O mixture. An amino acid was isolated from the polysaccharide by acid hydrolysis and identified as N(ε)-[(R)-1-carboxyethyl]-L-lysine ("alaninolysine", 2S,8R-alaLys) by determination of the specific optical rotation and (13)C NMR spectroscopy, using the authentic synthetic diastereomers 2S,8R-alaLys and 2S,8S-alaLys for comparison. The structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established.

[Bacterial antigen polysaccharides. 43. Structure of O-specific polysaccharide of Providencia alcalifaciens O46].

Acid O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. Consideration of the data revealed the following structure of the hexasaccharide repeating unit of O-specific polysaccharide under methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H, (1)H-COSY, TOCSY-, ROESY-,(1)H, (13)C-HSQC-, and HMQC-TOCSY experiments: [Formula: see text].

Pleiotropic effects of the lpxM mutation in Yersinia pestis resulting in modification of the biosynthesis of major immunoreactive antigens.

Deletion mutants in the lpxM gene in two Yersinia pestis strains, the live Russian vaccine strain EV NIIEG and a fully virulent strain, 231, synthesise a less toxic penta-acylated lipopolysaccharide (LPS). Analysis of these mutants revealed they possessed marked reductions in expression and immunoreactivity of numerous major proteins and carbohydrate antigens, including F1, Pla, Ymt, V antigen, LPS, and ECA. Moreover, both mutants demonstrated altered epitope specificities of the antigens as determined in immunodot-ELISAs and immunoblotting analyses using a panel of monoclonal antibodies. The strains also differed in their susceptibility to the diagnostic plague bacteriophage L-413C. These findings indicate that the effects of the lpxM mutation on reduced virulence and enhanced immunity of the Y. pestis EV DeltalpxM is also associated with these pleiotropic changes and not just to changes in the lipid A acylation.

A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs.

The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.

Full structure of the lipopolysaccharide of Pseudomonas aeruginosa immunotype 5.

The lipopolysaccharide (LPS) of the opportunistic human pathogen Pseudomonas aeruginosa immunotype 5 was delipidated by mild acid hydrolysis, and the products were separated by high-performance anion-exchange chromatography and analyzed by ESI MS and NMR spectroscopy. LPS species of three types were found, including those with an unsubstituted core and the core substituted with one O-polysaccharide repeating unit or with an O-polysaccharide of a variable number of repeating units. The core region is highly phosphorylated, the major species containing two monophosphate groups and one ethanolamine diphosphate group. Based on these and published data on the O-polysaccharide structure, the full structure of the LPS of P. aeruginosa immunotype 5 was established.

D- and L-aspartic acids: new non-sugar components of bacterial polysaccharides.

For the first time in bacterial polysaccharides, residues of D- and L-aspartic acids were identified as N-acyl substituents of 4-amino-4,6-dideoxy-D-glucose in the O-antigens of enterobacteria of the genera Providencia and Proteus.

[Structural diversity of O-polysaccharides and serological classification of Pseudomonas syringae pv. garcae and other strains of genomospecies 4]. / Strukturnoe raznoobrazie O-polisakharidov i serologicheskaia klassifikatsiia Pseudomonas syringae pv. garcae i drugikh shtammov genomotipa 4.

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I: --> 3)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 -->; II: --> 3)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 -->. The branched polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588 have the same L-rhamnan backbone with repeating units I and II and a lateral chain of (alpha1 --> 4)- or (alpha1 --> 3)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral alpha-D-Fuc3NAc residues were characterized.

Elucidation of the structure of the lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype 5 using strong alkaline degradation of the lipopolysaccharide.

The products of the strong alkaline degradation of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 5 were separated by anion-exchange HPLC and studied by electrospray ionization mass spectrometry and NMR spectroscopy. It was found that two major products have the same inner core region and lipid A carbohydrate backbone (A) but different outer core regions (B and C). The difference is in the position of a rhamnose residue, which is substituted with either an additional glucose residue (B) or a disaccharide remainder of the degraded O-polysaccharide (C). The site and the configuration of the linkage between the O-polysaccharide and the core were determined and, together with published data, the structure of the so-called biological repeating unit of the O-antigen was defined (D). The glycosidic linkage of the quinovosamine residue is beta when it links the O-polysaccharide to the core (C) and alpha when it connects the interior repeating units of the O-polysaccharide to each other (D) [Formula: see text]. In the structures shown Rha stands for rhamnose, Kdo for 3-deoxy-D-manno-oct-2-ulosonic acid, Hep for L-glycero-D-manno-heptose, GalNAcA for 2-acetamido-2-deoxygalacturonic acid, QuiN for 2-amino-2,6-dideoxyglucose (quinovosamine), DeltaHexNA for 2-amino-2-deoxy-D-threo-hex-4-enuronic acid; all monosaccharides are in the pyranose form and have the D configuration, except for Rha and GalNAcA that have the L configuration. In C, the remainder of the degraded O-polysaccharide is shown in bold type.

Structure of the O-polysaccharide of Pseudomonas syringae pv. delphinii NCPPB 1879(T) having side chains of 3-acetamido-3,6-dideoxy-D-galactose residues.

The O-polysaccharide (OPS) was obtained from the lipopolysaccharide of Pseudomonas syringae pv. delphinii NCPPB 1879(T) and studied by sugar and methylation analyses, Smith degradation, and (1)H- and (13)C-NMR spectroscopy. The OPS was found to contain residues of L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), and the following structure of the major (n = 2) and minor (n = 3) heptasaccharide repeating units of the OPS was established: [carbohydrate structure: see text]. The OPS is distinguished by the presence of oligosaccharide side chains consisting of three D-Fuc3NAc residues that are connected to each other by the (alpha 1-->2)-linkage. The OPS is characterized by a structural heterogeneity due to a different position of substitution of one of the four L-rhamnose residues in the main chain of the repeating unit as well as to the presence of oligosaccharide units with an incomplete side chain.

Identification of a homopolymer of 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-nonulosonic acid in the lipopolysaccharides of Legionella pneumophila Non-1 serogroups.

O-Specific polysaccharides (OPS) were isolated by mild acid hydrolysis of the lipopolysaccharides (LPS) of strains of Legionella pneumophila serogroups 2-14, as well as strains Lansing 3 and 16453-92 from newly proposed serogroups. The OPS were studied by (1)H- and (13)C-NMR spectroscopy, GLC/mass spectrometry, and chemical modifications (mild alkaline O-deacetylation and conversion of the N-acetimidoyl group into the N-acetyl group). All OPS were found to be a homopolymer of a 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxynonulosonic acid, which in some strains is 8-O-acetylated. In most strains studied, the monosaccharide has the D-glycero-D-talo configuration and is thus the C4 epimer of legionaminic acid (4-epilegionaminic acid), which has been previously identified as the monomer in the OPS of L. pneumophila serogroup 1. Poly(4-epilegionaminic acid) occurs as a minor polysaccharide in serogroups 5 (strain Dallas 1) and 13 and is absent in serogroups 1 and 7. The chemical basis for serological differentiation of L. pneumophila strains is discussed.

Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa.

Lipopolysaccharide (LPS) expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis patients lacks the O-polysaccharide chain but the degree to which the rest of the molecule changes has not been determined. We analyzed, for the first time, the core structure of an LPS from a rough, cystic fibrosis isolate of P. aeruginosa. The products of mild acid hydrolysis and strong alkaline degradation of the LPS were studied by ESI MS, MALDI MS, and NMR spectroscopy. The following structure was determined for the highest-phosphorylated core-lipid A backbone oligosaccharide isolated after alkaline deacylation of the LPS: [structure: see text] where Kdo and Hep are 3-deoxy-D-manno-octulosonic acid and L-glycero-D-manno-heptose, respectively; all sugars are in the pyranose form and have the D configuration unless stated otherwise. The outer core region occurs as two isomeric glycoforms differing in the position of rhamnose (Rha). The inner core region carries four phosphorylation sites at two Hep residues, HepI being predominantly bisphosphorylated and HepII monophosphorylated. In the intact LPS, both Hep residues carry monophosphate and diphosphate groups in nonstoichiometric quantities, GalN is N-acylated by an L-alanyl group, HepII is 7-O-carbamoylated, and the outer core region is nonstoichiometrically O-acetylated at four sites. Therefore, the switch to the LPS-rough phenotype in cystic fibrosis isolates of P. aeruginosa is not accompanied by losses of core monosaccharide, phosphate or acyl components. The exact positions of the O-acetyl groups and the role of the previously undescribed O-acetylation in the LPS core of P. aeruginosa remain to be determined.

Structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: (struture: see text). The main D-mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.

Structure of the O-specific polysaccharide isolated from the lipopolysaccharide of Citrobacter gillenii serotype O12a, 12b strain PCM 1544.

A neutral O-specific polysaccharide was isolated from the lipopolysaccharide of Citrobacter gillenii strain PCM 1544, representing serotype O12a,12b. The polysaccharide was studied by sugar and methylation analyses and Smith degradation along with 1H and 13C NMR spectroscopy, including a ROESY experiment. The following structure of the tetrasaccharide repeating unit was established, in which substitution with terminal GlcNAc is approximately 60%. [structure: see text]

Characterization of the lipopolysaccharide and structure of the O-specific polysaccharide of the bacterium Pseudomonas syringae pv. atrofaciens IMV 948.

Lipopolysaccharide (LPS) was isolated from the phytopathogenic bacterium Pseudomonas syringae pv. atrofaciens IMV 948 by mild extraction of the microbial cells with saline, and the properties, composition, and structure of the LPS were studied. The LPS showed low toxicity in D- galactosamine-sensitized mice and low biological activity in plants. Structural components of LPS--lipid A, core oligosaccharide, and O-specific polysaccharide (OPS)--were obtained by mild acid degradation and characterized. The lipid A contained fatty acids 3-HO-C10:0, C12:0, 2-HO-C12:0, 3-HO-C12:0, C16:0, C16:1, C18:0, and C18:1, as well as components of the hydrophilic moiety: GlcN, ethanolamine, phosphate, and phosphoethanolamine. The LPS core contained components typical of pseudomonads: glucose, rhamnose (Rha), L-glycero-D-manno-heptose, GlcN, GalN, 2-keto-3-deoxy-D-manno-octonic acid, alanine, and phosphate. The OPS consisted of L-Rha and D-GlcNAc in the ratio 4 : 1 and was structurally heterogeneous. The main pentasaccharide repeating unit of the OPS has the following structure: [structure see text]. Immunochemical studies showed that P. syringae pv. atrofaciens IMV 948 is serologically separate from other P. syringae strains, including those that have structurally similar OPS.

Structure of the O-specific polysaccharide of Vibrio cholerae O9 containing 2-acetamido-2-deoxy-D-galacturonic acid.

The O-specific polysaccharide (OPS) was isolated by mild-acid degradation of the lipopolysaccharide of Vibrio cholerae O9 and studied by carboxyl reduction, sugar and methylation analyses, Smith degradation, and two-dimensional NMR spectroscopy, including COSY, TOCSY, NOESY, and H-detected 1H,(13)C HMQC experiments. The following structure of the pentasaccharide-repeating unit of the OPS was established:

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223.

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.

Location of the O-methyl groups in the O polysaccharide of Pseudomonas syringae pv. phaseolicola.

The O-methylation pattern of the O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. phaseolicola GSPB 1552 was revealed by methylation (CD3I) analysis, Smith degradation, and NMR spectroscopy. Together with the major O repeats consisting of D-rhamnopyranose (D-Rhap) and D-fucofuranose (D-Fucf), there are minor repeats (approximately 30%) containing 3-O-methyl-D-rhamnose (D-acofriose), which is 2-substituted in the interior repeats and occupies the terminal non-reducing end of the OPS. It was suggested that the methylated O repeats are linked to each other nearby the non-reducing end of the OPS and that the 'biological' O repeat of the OPS has the following structure: [molecular structure: see text].

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196.

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established: -->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Galp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-GlcpNAc-(1-->.

Structural studies of the O-specific polysaccharide of Vibrio cholerae O8 using solvolysis with triflic acid.

The O-specific polysaccharide (OPS) of Vibrio cholerae 08 was isolated by mild acid degradation of the lipopolysaccharide and studied by two-dimensional NMR spectroscopy, including NOESY and heteronuclear multiple-bond correlation (HMBC) experiments. The OPS was found to have a tetrasaccharide repeating unit with the following structure: --> 4)-beta-D-Glcp NAc3NAcylAN-(1 --> 4)-beta-D-Manp NAc3NAcAN-(1 --> 4)-alpha-L-Gulp NAc3NAcA-(1 --> 3) -beta-D-QuipNAc4NAc-(1 --> where QuiNAc4NAc is 2,4-diacetamido-2,4,6-trideoxyglucose, GlcNAc3NAcylAN is 2-acetamido-3-(N-formyl-L-alanyl)amino-2,3-dideoxyglucuronamide, ManNAc3NAcAN is 2,3-diacetamido-2,3-dideoxymannuronamide, and GulNAc3NAcA is 2,3-diacetamido-2,3-dideoxyguluronic acid. The OPS was stable towards acid hydrolysis and solvolysis with anhydrous hydrogen fluoride, but could be cleaved selectively with trifluoromethanesulfonic (triflic) acid by the glycosidic linkages of beta-QuiNAc4NAc and alpha-GulNAc3NAcA. The structures of the oligosaccharides obtained that were elucidated by electrospray ionization (ESI) MS and NMR spectroscopy, confirmed the OPS structure.