Shigella flexneri O-antigens revisited: final elucidation of the O-acetylation profiles and a survey of the O-antigen structure diversity.

Shigella flexneri is an important human pathogen causing shigellosis. Strains of S. flexneri are serologically heterogeneous and, based on O-antigens, are currently classified into 14 types. Structures of the O-antigens (O-polysaccharides) of S. flexneri have been under study since 1960s but some gaps still remained. In this work, using one- and two-dimensional (1) H- and (13) C-NMR spectroscopy, the O-polysaccharides of several S. flexneri types were reinvestigated, and their structures were either confirmed (types 2b, 3b, 3c, 5b, X) or amended in respect to the O-acetylation pattern (types 3a, Y, 6, 6a). As a result, the O-acetylation sites were defined in all O-polysaccharides that had not been studied in detail earlier, and the long story of S. flexneri type strain O-antigen structure elucidation is thus completed. New and published data on the S. flexneri O-antigen structures are summarized and discussed in view of serological and genetic relationships of the O-antigens within the Shigella group and between S. flexneri and Escherichia coli.

O-antigen structure and gene clusters of Escherichia coli O51 and Salmonellaenterica O57; another instance of identical O-antigens in the two species.

The O-polysaccharides were released by mild acid hydrolysis from the lipopolysaccharides of Escherichia coli O51 and Salmonella enterica O57 and found to possess the same structure, which was established by sugar analysis and 1D and 2D NMR spectroscopy: [formula in text]. The O-antigen gene clusters of E. coli O51 and S. enterica O57 were sequenced and found to contain the same genes with a high-level similarity. All genes expected for the synthesis of the O-antigen were identified based on their similarity to genes from available databases.

Structural and genetic relationships of two pairs of closely related O-antigens of Escherichia coli and Salmonella enterica: E. coli O11/S. enterica O16 and E. coli O21/S. enterica O38.

The O-antigen is a part of the lipopolysaccharide molecule present in the outer membrane of Gram-negative bacteria, and is essential for the full function of the microorganisms. Salmonella enterica and Escherichia coli are taxonomically closely related species. In this study, the O-antigen structures of S. enterica O16 and O38 and E. coli O11 were determined. Salmonella enterica O38 and E. coli O21 were found to have identical O-antigen structures, whereas S. enterica O16 and E. coli O11 had closely related structures, differing only in the presence of a lateral glucose residue and O-acetylation of a mannose residue in the former. The O-antigen gene clusters of S. enterica O16 and O38 and E. coli O11 were sequenced and analyzed together with that of E. coli O21 retrieved from the GenBank. Each S. enterica/E. coli pair was found to contain the same set of genes organized in the same manner and to share 56-78% overall DNA identity. These data suggest that the O-antigen gene clusters of each pair studied originated from a common ancestor. Thus, it has become evident that in the past, the degree of relatedness between the O-antigens of S. enterica and E. coli was underestimated.

Structure and gene cluster of the O-antigen of Escherichia coli O109; chemical and genetic evidences of the presence of L-RhaN3N derivatives in the O-antigens of E. coli O109 and O119.

O-antigen representing the O-polysaccharide chain of the lipopolysaccharide is the most variable constituent on the cell surface of Gram-negative bacteria and a player in their pathogenicity. The O-polysaccharide of Escherichia coli O109 was studied by sugar analysis and nuclear magnetic resonance spectroscopy and found to contain a rarely occurring monosaccharide, 2,3-diacetamido-2,3,6-trideoxy-l-mannose (l-RhaNAc3NAc). The following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established, which is closely related to that of Proteus penneri O66: Ac--4-ß-L-RhapNAc3NAc -->4)-α-D-Glcp-(1-->3)-α-L-6dTalp-(1-->3)-ß-D-GlcpNAc-(1-->. The O-antigen gene cluster of E. coli O109 was sequenced and all 14 genes found were assigned functions based on their similarity to genes from the available databases. Putative genes for synthesis of l-RhaN3N were found in E. coli O109 and their homologues in E. coli O119, whose O-antigen has been reported earlier to contain 2-acetamido-2,3,6-trideoxy-3-formamido-d-mannose (d-RhaNAc3NFo). Analysis by GLC of the (S)-2-octyl glycosides confirmed that the absolute configuration of RhaN3N in E. coli O119 should be revised from D TO L.

Structural and genetic relationships between the O-antigens of Escherichia coli O118 and O151.

O-antigen (O-polysaccharide) is a highly variable part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which is used as the basis for bacterial serotyping and is essential for the full function and virulence of bacteria. In this work, the structure and genetics of the O-antigens of Escherichia coli O118 and O151 were investigated. Both O-polysaccharides were found to contain ribitol phosphate and have similar structures, the only difference between their backbones being one linkage mode (ß1→3 in E. coli O118 vs. ß1→2 in E. coli O151), which, most probably, is the linkage between the oligosaccharide repeats (O-units). The O-antigen gene clusters of the two bacteria are organized in the same manner and share high-level identity (>99%). Analysis of the wzy genes from E. coli O118 and O151 strains, which are responsible for the linkage between O-units, revealed only one nucleotide substitution, resulting in one amino acid residue substitution. The possible genetic events that may lead to the structural difference between two O-antigen structures are discussed. Salmonella O47 has the same O-unit backbone and a similar O-antigen gene cluster (OGC) (the DNA identity ranges from 74% to 83%) as E. coli O118 and O151. It was suggested that the OGCs of the three bacteria studied originated from a common ancestor.

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 (1)H and (13)C NMR spectroscopy: -->2)-alpha-l-Fucp-(1-->2)-beta-d-Galp-(1-->3)-alpha-d-GalpNAc-(1-->3)-alpha-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)-beta-d-Quip4NSerAc-(1-->3)-beta-d-Ribf-(1-->4)-alpha-d-GalpNAc-(1-->3)-alpha-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).

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: The O-antigen gene cluster of E. coli O161 was sequenced. In addition to the genes encoding sugar transferases, O-repeating unit flippase (Wzx) and O-antigen polymerase (Wzy), the genes involved in the biosynthesis of a legionaminic acid derivative were identified based on database similarities.

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 (1)H and (13)C NMR spectroscopy. They were found to possess a common -->2)-l-Rha-(alpha1-->2)-l-Rha-(alpha1-->3)-l-Rha-(alpha1-->3)-d-GlcNAc-(beta1--> 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 evidence for the close relationship between Escherichia coli O71 and Salmonella enterica O28 O-antigens.

O-antigen is the most variable cell wall constituent of Gram-negative bacteria. Escherichia coli and Salmonella enterica are closely related species. In this work, we present structural and genetic evidence for the close relationship between O-antigens of E. coli O71 and S. enterica O28. The E. coli O71 O-antigen was found to consist of tetrasaccharide-repeating units containing d-GalpNAc, d-Galp, l-Rhap, and d-Quip3NAc, with multiple O-acetyl lateral groups. It is very similar to the known structure of the S. enterica O28 O-antigen, which has the same backbone units, but with a lateral Glc residue instead of O-acetyl groups. The O-antigen gene clusters of E. coli O71 and S. enterica O28 were sequenced and found to contain the same genes with high-level similarity. All of the genes expected for the synthesis of the common backbone structure of the two O-antigens were identified based on homology. It is proposed that the two gene clusters had originated from the same ancestor, and diverged by acquiring prophage genes to carry out side-chain modifications. This is a new pair of the closely related E. coli and S. enterica O-serogroups. The serogroup-specific genes of E. coli O71 and S. enterica O28 were also identified.

Genetic and structural relationships of Salmonella O55 and Escherichia coli O103 O-antigens and identification of a 3-hydroxybutanoyltransferase gene involved in the synthesis of a Fuc3N derivative.

O-antigen (O-polysaccharide), a part of the outer membrane of Gram-negative bacteria, is one of the most variable cell constituents and is related to bacterial virulence. O-antigen diversity is almost entirely due to genetic variations in O-antigen gene clusters. In this study, the O-polysaccharide structures of Salmonella O55 and Escherichia coli O103 were elucidated by chemical analysis and nuclear magnetic resonance spectroscopy. It was found that the O-polysaccharides have similar pentasaccharide O-units, which differ only in one sugar (glucose versus N-acetylglucosamine) and in the N-acyl group (acetyl versus 3-hydroxybutanoyl) on 3-amino-3,6-dideoxy-d-galactose (d-Fuc3N). The Salmonella O55 antigen gene cluster was sequenced and compared with the E. coli O103 antigen gene cluster reported previously. The two gene clusters were found to share high-level similarity (DNA identity ranges from 53% to 76%), except for two putative acyl transferase genes (fdtC in Salmonella O55 and fdhC in E. coli O103) which show no similarity. Replacement of the fdtC gene in Salmonella O55 with the fdhC gene from E. coli O103 resulted in production of a modified O-antigen, which contains a 3-hydroxybutanoyl derivative of Fuc3N in place of 3-acetamido-3,6-dideoxygalactose. This finding strongly suggests that fdhC is a 3-hydroxybutanoyltransferase gene. The sequence similarity level suggested that the O-antigen gene clusters of Salmonella O55 and E. coli O103 originate from a common ancestor, and this evolutionary relationship is discussed.

Relatedness of the O-polysaccharide structures of Escherichia coli O123 and Salmonella enterica O58, both containing 4,6-dideoxy-4-{N-[(S)-3-hydroxybutanoyl]-D-alanyl}amino-D-glucose; revision of the E. coli O123 O-polysaccharide structure.

O-Polysaccharides were isolated by mild acid degradation of the lipopolysaccharides of Escherichia coli O123 and Salmonella enterica O58 and studied by chemical methods and 2D (1)H and (13)C NMR spectroscopy, including experiments in a H(2)O/D(2)O mixture, which enabled observation of correlations for nitrogen-linked protons. The following structure of the O-polysaccharide of E. coli O123 was established: -->3)-beta-D-Quip4NAlaHb-(1-->6)-alpha-D-GlcpNAc-(1-->3)-alpha-L-QuipNAc-(1-->3)-alpha-D-Glcp (6)(approx. 30% OAc)NAc-(1--> where L-QuipNAc stands for 2-acetamido-2,6-dideoxy-L-glucose and D-Qui4NAlaHb for 4-{N-[(S)-3-hydroxybutanoyl]-D-alanyl}amino-4,6-dideoxy-D-glucose. The latter was isolated as an ethylene glycol glycoside by three sequential Smith degradations of the O-deacetylated O-polysaccharide. The structure established in this work is at variance with the E. coli O123-polysaccharide structure reported earlier [Clark, C. G.; Kropinski, A. M.; Parolis, H.; Grant, C. C.; Trout-Yakel, K. M.; Franklin, K.; Ng, L. K.; Paramonov, N. A.; Parolis, L. A.; Rahn, K.; Tabor, H. J. Med. Microbiol.2009, 58, 884-894]. In accordance with the genetic data, the O-polysaccharide of S. enterica O58 has the same structure, except for it lacks the O-acetylation.

Structural and genetic characterization of Escherichia coli O99 antigen.

O-antigen is part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, and contributes the major antigenic variability to the cell surface. Screening for the Escherichia coli O-serogroup is the conventional method for identifying E. coli clones. In this study, we investigated the structural characteristics of the E. coli O99 O-antigen and the organization of the genes involved in its synthesis. On the basis of sugar and methylation analysis and nuclear magnetic resonance spectroscopy data, we established the structure of the branched hexasaccharide repeat unit of the O-polysaccharide. This unit consists of four d-rhamnose (d-Rha) moieties in the backbone and two d-glucose (d-Glc) moieties in the side chain, as shown below: [carbohydrate structure: see text]. The O-antigen gene cluster of E. coli O99, which was located between galF and gnd, was found to contain putative genes for the synthesis of d-Rha, genes encoding sugar transferases, and ATP-binding cassette (ABC) transporter genes (wzm and wzt). Our findings indicate that in E. coli O99, the synthesis and translocation of the O-antigen occurs by an ABC transporter-dependent process.

A group of Escherichia coli and Salmonella enterica O antigens sharing a common backbone structure.

The O-antigen moiety of the LPS is one of the most variable cell surface components of the Gram-negative bacterial outer membrane. Variation is due to the presence of different sugars and sugar linkages. Here, it is reported that a group of Escherichia coli O serogroups (O17, O44, O73, O77 and O106), and the Salmonella enterica serogroup O : 6,14 (H), share a common four-sugar backbone O-subunit structure, and possess almost identical O-antigen gene clusters. Whereas the E. coli O77 antigen does not have any substitutions, the other O antigens in this group differ by the addition of one or two glucose side branches at various positions of the backbone. The O-antigen gene clusters for all members of the group encode only the proteins required for biosynthesis of the common four-sugar backbone. The identification of three genes within a putative prophage in the E. coli O44 genome is also reported; these genes are presumably involved in the glucosylation of the basic tetrasaccharide unit. This was confirmed by deletion of one of the genes, which encodes a putative glucosyltransferase. Structural analysis of the O antigen produced by the mutant strain demonstrated the absence of glucosylation. An O-antigen structure shared by five E. coli and one S. enterica serogroups, all of which have a long evolutionary history, suggests that the common backbone may be important for the survival of E. coli strains in the environment, or for their pathogenicity.

Structural and genetic evidence that the Escherichia coli O148 O antigen is the precursor of the Shigella dysenteriae type 1 O antigen and identification of a glucosyltransferase gene.

Shigella dysenteriae type 1 is the most virulent serotype of Shigella. Enterotoxigenic Escherichia coli O148 is pathogenic and can cause diarrhoea. The following structure was established for the tetrasaccharide repeating unit of the E. coli O148 O antigen: -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpNAc-(1-->. This differs from the structure reported earlier for S. dysenteriae type 1 by having a glucose (Glc) residue in place of a galactose (Gal) residue. The two bacteria also have the same genes for O antigen synthesis, with the same organization and high level of DNA identity, except that in S. dysenteriae type 1 wbbG is interrupted by a deletion, and a galactosyltransferase gene wbbP located on a plasmid is responsible for the transfer of galactose to make a novel antigenic epitope of the O antigen. The S. dysenteriae type 1 O antigen was reconstructed by replacing the E. coli O148 wbbG gene with the wbbP gene, and it had the LPS structure and antigenic properties of S. dysenteriae type 1, indicating that the S. dysenteriae type 1 O antigen evolved from that of E. coli O148. It was also confirmed that wbbG of E. coli O148 is a glucosyltransferase gene, and two serotype-specific genes of E. coli O148 and S. dysenteriae type 1 were identified.

Structure of the O-polysaccharide of Escherichia coli O150 containing 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxy-d-glucose.

An acidic O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Escherichia coli O150 and studied by sugar and methylation analyses, triflic acid solvolysis, Smith degradation, (1)H and (13)C NMR spectroscopy, including 2D ROESY, (1)H,(13)C HSQC, HMQC-TOCSY, and HMBC experiments. The polysaccharide was found to contain a regioisomer of N-acetylisomuramic acid, 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxy-d-glucose [d-GlcNAc4(Slac)]. The structure of its hexasaccharide repeating unit was established.

Structural and genetic characterization of Shigella boydii type 17 O antigen and confirmation of two new genes involved in the synthesis of glucolactilic acid.

Shigella strains are human pathogens and normally identified based on their O antigens. The chemical structure and gene cluster of Shigella boydii type 17 O antigen were studied. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen of S. boydii type 17 has a linear trisaccharide O unit, which consists of two residues of N-acetylgalactosamine (GalNAc) and a 4-O-[(R)-1-carboxyethyl]-d-glucose (glucolactilic acid). The O antigen gene cluster of S. boydii type 17 was sequenced and genes encoding UDP-N-acetylglucosamine C4 epimerase for GalNAc synthesis, O unit flippase, O antigen polymerase, and glycosyltransferases were putatively identified based on sequence similarities and the presence of conserved motifs. Two genes, whose functions could not be clearly indicated by homology search, were confirmed to be involved in the synthesis of glucolactilic acid by mutation and structural verification of the O antigens from the mutants. To our knowledge, this is the first time that genes involved in the synthesis of glucolactilic acid have been reported. Two genes specific to S. boydii type 17 were also identified.

Structural and molecular characterization of Shigella boydii type 16 O antigen.

Shigella is a well-known human pathogen causing dysentery and their typing is solely based on the O antigens. We investigated the chemical structure and gene cluster of Shigella boydii type 16 O antigen. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen has an O-acetylated branched pentasaccharide repeating O unit, which consists of two D-mannose residues (D-Man), one residue each of d-glucuronic acid (D-GlcA), N-acetylglucosamine (D-GlcNAc) and D-galactose (D-Gal), and the structure of the O unit was established. The O antigen gene cluster of S. boydii type 16 was identified and shown to contain putative genes for the synthesis of GDP-D-Man, genes encoding sugar transferases, O unit flippase (Wzx) and O antigen polymerase (Wzy) as expected. The function of the wzy gene was characterized by mutation test. Genes specific to S. boydii type 16 O antigen gene cluster were identified by screening 186 Escherichia coli and Shigella type strains, and can be used to develop PCR assays for detection of type 16 strains.

Structure of a teichoic acid-like O-polysaccharide of Escherichia coli O29.

A teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O29. The O-polysaccharide and an oligosaccharide obtained by dephosphorylation of the O-polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The following structure of the branched oligosaccharide repeating unit, containing five monosaccharide residues and glycerol 1-phosphate (D-Gro-1-P), was established: [carbohydrate structure: see text].

Structural and genetic characterization of the Shigella boydii type 18 O antigen.

Shigella strains are important human pathogens and are normally identified by their O antigens. O antigen is an essential part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria and plays a role in pathogenicity. Structural and genetic organization of the Shigella boydii type 18 O antigen was investigated. As judged by sugar and methylation analyses and NMR spectroscopy data, the O antigen has a linear pentasaccharide repeating unit (O unit), which consists of three L-rhamnose residues, and one residue each of D-galacturonic acid (D-GalA) and N-acetylgalactosamine (D-GalNAc), and the following structure of the O unit was established. -->3)-beta-L-Rhap-(1-->4)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-GalpA-(1-->3)-alpha-D-GalpNAc-(1--> The O antigen gene cluster of S. boydii type 18, which contains nine open reading frames (ORFs), was found between galF and gnd. Based on homology, all of the ORFs were identified as O antigen synthesis genes, involved in the synthesis of rhamnose, transfer of sugars, and processing of O unit. Genes specific for S. boydii type 18 were identified, which can be potentially used for the development of a PCR-based assay for the identification and detection of this strain.