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.     

A new ethanolamine phosphate-containing variant of the O-antigen of Shigella flexneri type 4a

The O-specific polysaccharide (O-antigen) structure of a Shigella flexneri type 4a strain from the Dysentery Reference Laboratory (London, UK) was elucidated in 1978 and its characteristic feature was found to be α-d-glucosylation of GlcNAc at position 6, which defines O-factor IV. Our NMR spectroscopic studies of the O-specific polysaccharides of two other strains belonging to S. flexneri type 4a (G1668 from Adelaide, Australia, and 1359 from Moscow, Russia) confirmed the carbohydrate backbone structure but revealed in both strains an additional component, ethanolamine phosphate (EtnP), attached at position 3 of one of the rhamnose residues:

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Phosphorylation has not been hitherto reported in any S. flexneri O-antigen. Reinvestigation of the O-specific polysaccharide of S. flexneri type 4b showed that it is not phosphorylated and confirmed its structure established earlier.     

The O-polysaccharide of Escherichia coli O112ac has the same structure as that of Shigella dysenteriae type 2 but is devoid of O-acetylation: a revision of the S. dysenteriae type 2 O-polysaccharide structure

The O-antigen structure of Shigella dysenteriae type 2 was reinvestigated using chemical modifications along with high-resolution 2D (1)H and (13)C NMR spectroscopy. The O-antigen was found to contain a pyruvic acid acetal, which was overlooked in an early study, and the following revised structure of the pentasaccharide repeating unit was established: where approximately 70% GlcNAc residues bear an O-acetyl group at position 3. The O-antigen of Escherichia coli O112ac was found to have the same carbohydrate structure but to lack O-acetylation.     

Structure of the O-polysaccharide of Cronobacter sakazakii O2 with a randomly O-acetylated l-rhamnose residue

Studies by sugar analysis and partial acid hydrolysis along with one- and two-dimensional ¹H and ¹³C NMR spectroscopy and high-resolution ESI MS showed that the O-polysaccharide (O-antigen) Cronobacter sakazakii ATCC 29004 (serotype O2) possesses a branched hexasaccharide O-unit with a randomly mono-O-acetylated terminal rhamnose residue in the side chain and the following structure:A similar structure has been reported for the O-polysaccharide of C. sakazakii 767, which differs in the presence of an additional lateral α-d-Glcp residue on GlcNAc and the pattern of O-acetylation (Czerwicka, M., Forsythe, S. J.; Bychowska, A.; Dziadziuszko, H.; Kunikowska, D.; Stepnowski, P.; Kaczynski, Z. Carbohydr. Res.2010, 345, 908-913).     

Structure of the O-antigen of Yersinia pseudotuberculosis O:4a revised

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Yersinia pseudotuberculosis O:4a and studied by NMR spectroscopy, including 2D ROESY and ¹H, ¹³C HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established, which differs from the structure reported earlier [Gorshkova, R. P. et al., Bioorg. Khim. 1983, 9, 1401-1407] in the linkage modes between the monosaccharides: where Tyv stands for 3,6-dideoxy-d-arabino-hexose (tyvelose). The structure of the Y. pseudotuberculosis O:4a antigen resembles that of Y. pseudotuberculosis O:2c, which differs in the presence of abequose (3,6-dideoxy-d-xylo-hexose) in place of tyvelose only.     

Reinvestigation of the O-antigens of Yersinia pseudotuberculosis: revision of the O2c and confirmation of the O3 antigen structures

Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: where Abe stands for 3,6-dideoxy-d-xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

Close relation of the O-polysaccharide structure of Escherichia coli O168 and revised structure of the O-polysaccharide of Shigella dysenteriae type 4

The O-polysaccharide was isolated from the lipopolysaccharide of Escherichia coli O168 and studied by chemical analyses and Smith degradation along with (1)H and (13)C NMR spectroscopies. The following structure of the branched pentasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text] where 6-O-acetylation of GlcNAc is partial. Reinvestigation of the O-polysaccharide of Shigella dysenteriae type 4 established earlier showed it to have the same structure except for that the lateral Fuc residue is nonstoichiometrically O-acetylated at each position.     

Elucidation of the structure and characterization of the gene cluster of the O-antigen of Cronobacter sakazakii G2592, the reference strain of C. sakazakii O7 serotype

The O-specific polysaccharide from the lipopolysaccharide of Cronobacter sakazakii G2592 was studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following structure of the pentasaccharide repeating unit was established:This structure is unique among the known bacterial polysaccharide structures, which is in accord with classification of strain G2592 into a new C. sakazakii serotype, O7. It is in agreement with the O-antigen gene cluster of this strain, which was found between the housekeeping genes JUMPStart and gnd and characterized by sequencing and tentative assignment of the gene functions.     

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 of the O-polysaccharide of Providencia alcalifaciens O8 containing (2S,4R)-2,4-dihydroxypentanoic acid, a new non-sugar component of bacterial glycans

A glycerol teichoic acid-like O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O8 and studied by chemical methods and NMR spectroscopy, including 2D ROESY, {(1)H,(13)C} HSQC, and HMQC-TOCSY experiments. It was found that the compound contains a new component of bacterial lipopolysaccharides: ether-linked (2S,4R)-2,4-dihydroxypentanoic acid (Dhpa), which was identified by NMR spectroscopy. The following structure of the repeating unit of the polysaccharide was established:     

Activating mutations of N-WASP alter Shigella pathogenesis

The pathogenesis of Shigella requires binding to the host protein N-WASP. To examine the roles of structural conformation and phospho-regulation of N-WASP during Shigella pathogenesis, mutant N-WASP constructs predicted to result in a constitutively open conformation (L229P and L232P) or either a phospho-mimicking (Y253E) or phospho-disruptive (Y253F) structure were constructed. Pyrene actin assays demonstrated that the N-WASP L229P and L232P constructs are constitutively active. Despite the increase in actin polymerization seen in vitro, cell lines expressing N-WASP L229P and L232P supported shorter actin tails when infected with Shigella.Shigella actin tails were unchanged in cells expressing N-WASP phospho-regulation mutant proteins. Shigella invasion, intracellular, and intercellular motility were not altered in cells expressing N-WASP L229P or L232P. However, plaque numbers were increased in cells expressing N-WASP L229P and L232P. These data demonstrate that N-WASP structural conformation is an important regulator of Shigella pathogenesis in distinct segments of its lifecycle.     

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 the lipopolysaccharide of Rahnella aquatilis 1-95

The O-polysaccharide was isolated by mild acid hydrolysis of the lipopolysaccharide of Rahnella aquatilis 1-95 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including NOESY and 1H,13C HSQC experiments for linkage and sequence analysis. The following structure of the branched trisaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text].