Structure of the O-polysaccharide of Salmonella enterica O41

An O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Salmonella enterica O41, and the following structure of the O-unit was determined by chemical analyses along with 1D and 2D ¹H and ¹³C NMR spectroscopy:→2)-β-d-Manp-(1→4)-α-d-Glcp-(1→3)-α-l-QuipNAc-(1→3)-α-d-GlcpNAc-(1→where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose. The structure established is in agreement with the O-antigen gene cluster of S. enterica O41 and tentative assignment of the gene functions reported earlier.     

Structures of the O-polysaccharides of Salmonella enterica O59 and Escherichia coli O15

The O-polysaccharide of Salmonella enterica O59 was studied using sugar analysis and 2D ¹H and ¹³C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit was established:→2)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→4)-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→Accordingly, the O-antigen gene cluster of S. enterica O59 includes all genes necessary for the synthesis of this O-polysaccharide. Earlier, another structure has been reported for the O-polysaccharide of Salmonella arizonae (S. enterica IIIb) O59, which later was found to be identical to that of Citrobacter (Citrobacter braakii) O35 and, in this work, also to the O-polysaccharide of Escherichia coli O15.     

The O-antigen of Salmonella enterica O13 and its relation to the O-antigen of Escherichia coli O127

The following structure of the O-polysaccharide (O-antigen) of Salmonella enterica O13 was established by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:→2)-α-l-Fucp-(1→2)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen of S. enterica O13 was found to be closely related to that of Escherichia coli O127, which differs only in the presence of a GalNAc residue in place of the GlcNAc residue and O-acetylation. The location of the O-acetyl groups in the E. coli O127 polysaccharide was determined. The structures of the O-polysaccharides studied are in agreement with the DNA sequence of the O-antigen gene clusters of S. enterica O13 and E. coli O127 reported earlier.     

Structural and genetic characterization of the O-antigen of Salmonella enterica O56 containing a novel derivative of 4-amino-4,6-dideoxy-d-glucose

Based on the O-antigens (O-polysaccharides), one of the most variable cell constituents, 46 O-serogroups have been recognized in the Kauffmann-White serotyping scheme for Salmonella enterica. In this work, the structure of the O-polysaccharide and the genetic organization of the O-antigen gene cluster of S. enterica O56 were investigated. As judged by sugar and methylation analyses, along with NMR spectroscopic data, the O-polysaccharide has a linear tetrasaccharide O-unit, which consists of one residue each of d-ribofuranose, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, and a novel sugar derivative, 4-(N-acetyl-l-seryl)amino-4,6-dideoxy-d-glucose (d-Qui4NSerAc). The following structure of the O-polysaccharide was established:→3)-β-d-Quip4NSerAc-(1→3)-β-d-Ribf-(1→4)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen gene cluster of S. enterica O56 having 12 open reading frames was found between the housekeeping genes galF and gnd. A comparison with databases and using the O-antigen structure data enabled us to ascribe functions to genes for (i) synthesis of d-GalNAc and d-Qui4NSerAc, (ii) sugar transfer, and (iii) O-antigen processing, including genes for O-unit flippase (Wzx) and O-antigen polymerase (Wzy).     

Structure of the O-polysaccharide of Vibriocholerae O43 containing a new monosaccharide derivative, 4-(N-acetyl-l-allothreonyl)amino-4,6-dideoxy-d-glucose

The O-polysaccharide of Vibriocholerae O43 was studied using chemical analyses, triflic acid solvolysis and 2D NMR spectroscopy, including ¹H/¹H COSY, TOCSY, NOESY and ¹H/¹³C gradient-selected HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:→3)-β-d-Quip4NAcyl-(1→3)-α-d-GalpNAcA-(1→4)-α-d-GalpNAc-(1→3)-α-d-QuipNAc-(1→where d-QuiNAc stands for 2-acetamido-2,6-dideoxy-d-glucose, d-Qui4NAcyl for 4-(N-acetyl-l-allothreonyl)amino-4,6-dideoxy-d-glucose and d-GalNAcA for 2-acetamido-2-deoxy-d-galacturonic acid.     

Structural studies of the O-antigenic polysaccharide from Escherichia coli O177

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by ¹H,¹³C-heteronuclear multiple-bond correlation and ¹H,¹H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure:→2)-α-l-Rhap-(1→3)-α-l-FucpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→An α-l-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the ¹H and ¹³C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.     

The structural characterization of the O-polysaccharide antigen of the lipopolysaccharide of Escherichiacoli serotype O118 and its relation to the O-antigens of Escherichiacoli O151 and Salmonellaenterica O47

Mild acid hydrolysis of the lipopolysaccharide produced by Escherichiacoli O118:H16 standard strain (NRCC 6613) afforded an O-polysaccharide (O-PS) composed of d-galactose, 2-acetamidoylamino-2,6-dideoxy-l-galactose , 2-acetamido-2-deoxy-d-glucose, ribitol, and phosphate (1:1:1:1:1). From DOC-PAGE, sugar and methylation analyses, one- and two-dimensional NMR spectroscopy, capillary electrophoresis-mass spectrometry, hydrolysis, and sequential Smith-type periodate oxidation studies, the O-PS was determined to be an unbranched linear polymer having the structure:[6)-α-d-Galp-(1→3)-α-l-FucpNAm-(1→3)-β-d-GlcpNAc-(1→3)-Rib-ol-5-P-(O→]_nThe structure of the O-PS is consistent with the reported DNA data on the O-antigen gene-cluster of E. coli O118 and interestingly, the O-PS is similar to the structures of the O-antigens of Salmonellaenterica O47 and E. coli O151:H10 reference strain 880-67, as predicted from the results of DNA sequencing of their respective O-antigen gene-clusters.     

Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c)

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. ¹H and ¹³C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap2Ac-(1→3)[α-d-Glcp-(1→2)-α-d-Glcp-(1→4)]-β-d-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.     

Novel O-antigen of Hafnia alvei PCM 1195 lipopolysaccharide with a teichoic acid-like structure

The lipopolysaccharide (LPS) of Hafnia alvei strain PCM 1195 was obtained by the hot phenol/water method. The O-specific polysaccharide was released by mild acidic hydrolysis and isolated by gel filtration. The structure of the O-specific polysaccharide was investigated by ¹H, ¹³C, and ³¹P NMR spectroscopy, MALDI-TOF MS, and GC-MS, accompanied by monosaccharide and methylation analysis. It was concluded that the O-specific polysaccharide is composed of a hexasaccharide repeating units interlinked with a phosphate group: {→4-α-d-Glcp-(1→3)-α-l-FucpNAc-(1→3)-[α-d-Glcp-(1→4)]-α-d-GlcpNAc-(1→3)-α-l-FucpNAc-(1→4)-α-d-Glcp-(1→P}_n.     

A highly α-selective glycosylation for the convenient synthesis of repeating α-(1→4)-linked N-acetyl-galactosamine units

The repeating GalpNAc-α-(1→4)-GalpNAc unit is part of a series of essential structures that can be found in many important biomolecules such as the glycoproteins and the O-antigenic polysaccharides of clinically important bacterial strains. In this paper, we describe an exclusive α-selective glycosylation reaction, using a 4,6-di-O-tert-butyldimethylsilyl-N-acetyloxazolidinone-protected thioglycoside as the glycosyl donor, under pre-activation conditions, with only half amount of the promoter, providing the product GalpNAc-α-(1→4)-GalpNAc in high isolated yield. This reaction can be also applied to increasing the length of the repeating structure, which is of significant use in further synthesis of branched or linear oligosaccharides.     

Structural analysis of the O-specific polysaccharide isolated from Plesiomonas shigelloides O51 lipopolysaccharide

Plesiomonasshigelloides strain CNCTC 110/92 (O51) was identified as a new example of plesiomonads synthesising lipopolysaccharides (LPSs) that show preference for a non-aqueous surrounding during phenol/water extraction. Chemical analyses combined with ¹H and ¹³C NMR spectroscopy, MALDI-TOF and ESI mass spectrometry showed that the repeating units of the O-specific polysaccharides isolated from phenol and water phase LPSs of P. shigelloides O51 have the same structure: →4)-β-d-GlcpNAc3NRA-(1→4)-α-l-FucpAm3OAc-(1→3)-α-d-QuipNAc-(1→, containing the rare sugar constituent 2,3-diamino-2,3-dideoxyglucuronic acid (GlcpNAc3NRA), and substituents such as d-3-hydroxybutyric acid (R) and acetamidino group (Am). The HR-MAS NMR spectra obtained for the isolated LPSs and directly on bacteria indicated that the O-acetylation pattern was consistent throughout the entire preparation. The ¹H chemical shift values of the structure reporter groups identified in the isolated O-antigens matched those present in bacteria. We have found that the O-antigens recovered from the phenol phase showed a higher degree of polymerisation than those isolated from the water phase.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.     

Structure of the O-polysaccharide of Pragia fontium 27480 containing 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid

The following structure of the O-polysaccharide of Pragia fontium 27480 was elucidated by sugar analysis, including determination of the absolute configurations of the monosaccharides, and Smith degradation along with 1D and 2D ¹H and ¹³C NMR spectroscopy:→4)-β-d-ManpNAc3NAcA-(1→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→4)-α-d-GlcpNAc-(1→where ManNAc3NAcA stands for 2,3-diacetamido-2,3-dideoxymannuronic acid.     

Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O175

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O175 has been elucidated. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments were used to determine the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY, and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure:→2)-α-d-Glcp-(1→4)-α-d-GlcpA-(1→3)-α-d-Manp-(1→2)-α-d-Manp-(1→3)-β-d-GalpNAc-(1→Cross-peaks of low intensity from an α-linked glucopyranosyl residue were present in the ¹H,¹H-TOCSY NMR spectra. The α-d-Glcp residue is suggested to originate from the terminal part of the polysaccharide and consequently the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O175 O-antigen is similar to those from E. coli O22 and O83, both of which carry an α-d-Glcp-(1→4)-d-GlcpA structural element, thereby explaining the reported cross-reactivities between the strains.     

Structure and gene cluster of the O-antigen of Escherichia coli O19ab

The O-polysaccharide (O-antigen) of Escherichia coli O19ab was studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy. The following structure of the linear pentasaccharide repeating unit was established:→2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-d-Glcp-(1→3)-α-d-GlcpNAc6Ac-(1→where the degree of O-acetylation of GlcNAc is ∼33%. The O-antigen gene cluster of E. coli O19ab was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the E. coli O19ab-antigen structure.     

Structure of the O-polysaccharide of the lipopolysaccharide of Pragia fontium 97U116

The O-polysaccharide of Pragia fontium 97U116 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy. The following structure of the pentasaccharide-repeating unit was established: →2)-α-d-Galf-(1→3)-α-l-Rhap2Ac^I-(1→4)-α-d-GlcpNAc^I-(1→2)-α-l-Rhap^II-(1→3)-β-d-GlcpNAc^II-(1→     

Structure of a polysaccharide from the lipopolysaccharides of Vibrio vulnificus strains CECT 5198 and S3-I2-36, which is remarkably similar to the O-polysaccharide of Pseudoalteromonas rubra ATCC 29570

High-molecular-mass polysaccharides were released by mild acid degradation of the lipopolysaccharides of two wild-type Vibrio vulnificus strain, a flagellated motile strain CECT 5198 and a non-flagellated non-motile strain S3-I2-36. Studies by sugar analysis and partial acid hydrolysis along with ¹H and ¹³C NMR spectroscopies showed that the polysaccharides from both strains have the same trisaccharide repeating unit of the following structure:→4)-β-d-GlcpNAc3NAcylAN-(1→4)-α-l-GalpNAmA-(1→3)-α-d-QuipNAc-(1→where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose, GalNAmA for 2-acetimidoylamino-2-deoxygalacturonic acid, GlcNAc3NAcylAN for 2-acetamido-3-acylamino-2,3-dideoxyglucuronamide and acyl for 4-d-malyl (∼30%) or 2-O-acetyl-4-d-malyl (∼70%). The structure of the polysaccharide studied resembles much that of a marine bacterium Pseudoalteromonas rubra ATCC 29570 reinvestigated in this work. The latter differs in (i) the absolute configuration of malic acid (l vs d), (ii) 3-O-acetylation of GalNAmA and (iii) replacement of QuiNAc with its 4-keto biosynthetic precursor.     

Structures of the O-antigens of Escherichia coli O13, O129, and O135 related to the O-antigens of Shigella flexneri

O-Polysaccharides (O-antigens) were isolated from Escherichia coli O13, O129, and O135 and studied by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy. They were found to possess a common →2)-l-Rha-(α1→2)-l-Rha-(α1→3)-l-Rha-(α1→3)-d-GlcNAc-(β1→ backbone, which is a characteristic structural motif of the O-polysaccharides of Shigella flexneri types 1-5. In both the bacterial species, the backbone is decorated with lateral glucose residues or/and O-acetyl groups. In E. coli O13, a new site of glycosylation on 3-substituted Rha was revealed and the following O-polysaccharide structure was established:The structure of the E. coli O129 antigen was found to be identical to the O-antigen structure of S. flexneri type 5a specified in this work and that of E. coli O135 to S. flexneri type 4b reported earlier.     

Structural and genetic characterization of the O-antigen of Escherichia coli O161 containing a derivative of a higher acidic diamino sugar, legionaminic acid

The O-antigen is an essential component of lipopolysaccharide on the surface of Gram-negative bacteria and plays an important role in its pathogenicity. Composition and structure of the O-antigens of Escherichia coli are highly diverse mainly due to genetic variations in the O-antigen gene cluster. In this work, the chemical structure and the gene cluster of the O-antigen of E. coli O161 were studied. Chemical degradations, sugar analyses, and NMR spectroscopy showed that the O161 antigen possesses a trisaccharide O-repeating unit containing a 5-N-acetyl-7-N-(d-alanyl) derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7Ala) and having the following structure:

→8)-α-Legp5Ac7Ala-(2→4)-β-d-GlcpA-(1→3)-β-d-GlcpNAc-(1→     

Low structural diversity of the O-polysaccharides of Photorhabdus asymbiotica subspp. asymbiotica and australis and their similarity to the O-polysaccharides of taxonomically remote bacteria including Francisella tularensis

The O-polysaccharides were isolated from the lipopolysaccharides of emerging human pathogens Photorhabdus asymbiotica subsp. asymbiotica US-86 and US-87 and subsp. australis AU36, AU46, and AU92. Studies by sugar analysis and ¹H and ¹³C NMR spectroscopy before and after O-deacetylation showed that the O-polysaccharide structures are essentially identical within, and only slightly different between, the subspecies. The following structures of the repeating units of the O-polysaccharides were established:→3)-β-d-Quip4NGlyFo-(1→4)-α-d-GalpNAcAN3Ac-(1→4)-α-d-GalpNAcA3R-(1→3)-α-d-QuipNAc-(1→where GalNAcA stands for 2-acetamido-2-deoxygalacturonic acid, GalNAcAN for amide of GalNAcA, QuiNAc for 2-acetamido-2,6-dideoxyglucose, and Qui4NGlyFo for 4,6-dideoxy-4-(N-formylglycyl)aminoglucose; R = Ac in subsp. asymbiotica or H in subsp. australis. The structures established resemble those of a number of taxonomically remote bacteria including Francisella tularensis (Vinogradov, E. V.; Shashkov, A. S.; Knirel, Y. A.; Kochetkov, N. K.; Tochtamysheva, N. V.; Averin, S. P.; Goncharova, O. V.; Khlebnikov, V. S. Carbohydr. Res.1991, 214, 289–297), which differs in (i) the presence of a formyl group on Qui4N rather than the N-formylglycyl group, (ii) the mode of the linkage between the repeating units (β1→2 vs α1→3), (iii) amidation of both GalNAcA residues rather than one residue, and iv) the lack of O-acetylation.