Structures and genetics of Kdo-containing O-antigens of Cronobacter sakazakii G2706 and G2704, the reference strains of serotypes O5 and O6

Cronobacter sakazakii G2706 and G2704 are the reference strains of serotypes O5 and O6 in the serological classification of this species proposed recently. Mild acid degradation of the lipopolysaccharides of both strains resulted in cleavage of the O-polysaccharide chains at the acid-labile linkage of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to yield oligosaccharides representing repeating units of the O-polysaccharides. The oligosaccharides and alkali-degraded lipopolysaccharides were studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following O-polysaccharide structures were established:The structure of strain G2706 is unique among the known bacterial polysaccharide structures, whereas that of strain G2704 is identical to the structure of Cronobacter malonaticus 3267 [MacLean, L. L.; Vinogradov, E.; Pagotto, F.; Farber, J. M.; Perry, M. B. Biochem. Cell Biol.2009, 87, 927–932], except for that the latter lacks O-acetylation. Putative functions of the genes in the O-antigen gene clusters of C. sakazakii strains studied are in agreement with the O-polysaccharide structures.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as ¹H and ¹³C NMR spectroscopy, including 2D ROESY and ¹H,¹³C HMBC experiments in D₂O and a ROESY experiment in a 9:1 H₂O–D₂O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure:The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.     

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.     

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.     

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.     

Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O173

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O173 has been investigated. Sugar and methylation analyses, electrospray ionisation mass spectrometry together with ¹H, ³¹P and ¹³C NMR spectroscopy were the main methods used. The structure of the pentasaccharide repeating unit of the PS was found to be:

By treatment with 48% HF the phosphoric diester linkage was cleaved together with the glycosidic linkage of the fucosyl group, rendering a tetrasaccharide with the structure:

     

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.     

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).     

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: 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.     

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 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.     

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.     

Structure elucidation of the O-antigen of <Emphasis Type="Italic">Salmonella enterica</Emphasis> O51 and its structural and genetic relation to the O-antigen of <Emphasis Type="Italic">Escherichia coli</Emphasis> O23

The O-polysaccharide (O-antigen) of Salmonella enterica O51 was isolated by mild acid degradation of the lipopolysaccharide and its structure was established using sugar analysis and NMR spectroscopy. The O-antigen of Escherichia coli O23, whose structure was elucidated earlier, possesses a similar structure and differs only in the presence of an additional lateral α-D-Glcp residue at position 6 of the GlcNAc residue in the main chain. Sequencing of the O-antigen gene clusters of S. enterica O51 and E. coli O23 revealed the same genes with a high-level similarity. By comparison with opened gene databases, all genes expected for the synthesis of the common structure of the two O-antigens were assigned functions. It is suggested that the gene clusters of both bacteria originated from a common ancestor, whereas the O-antigen modification in E. coli O23, which, most probably, is induced by prophage genes outside the gene cluster, could be introduced after the species divergence.     

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.     

Synthesis of 2′,3′-Dideoxy-2′-fluoro-3′-thioarabinothymidine and Its 3′-Phosphoramidite Derivative

Abstract

An efficient method for the synthesis of 5′-O-monomethoxytrityl-2′,3′-dideoxy-2′-fluoro-3′-thioarabinothymidine [^5′-MMTaraF-T_3′SH, (5)] and its 3′-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5′-O-MMT-2,3′-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q., J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give ^5′-MMTaraF-T_3′SAc (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, Lönnberg, H. Eur. J. Org. Chem. 2000, 1987–1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352–1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141–2150; (c) Fettes, K.J.; O’Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351–1354).     

Structural relationships between genetically closely related O-antigens of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using ¹H and ¹³C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.     

Structural determination of the O-antigenic polysaccharide from Escherichia coli O74

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O74 has been determined. Component analysis, together with ¹H and ¹³C NMR spectroscopy as well as ¹H,¹⁵N-HSQC experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure:

Full-size image (5K)

Cross-peaks of low intensity from an α-linked N-acetylglucosamine residue were present in the NMR spectra, and spectral analysis indicates that they originate from the penultimate residue in the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The ¹H, ¹³C and ¹⁵N NMR chemical shifts of the α- and β-anomeric forms of d-Fucp3NAc are also reported. The repeating unit of the E. coli O74 O-antigen is identical to that of the capsular polysaccharide from E. coli K45.     

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.