New lactate- and pyruvate-containing polysaccharide and rhamnomannan with xylose residues from the cell wall of Rathayibacter oskolensis VKM Ac-2121T.

The cell wall of endophytic strain Rathayibacter oskolensis VKM Ac-2121T (family Microbacteriaceae, class Actinomycetes) was found to contain neutral and acidic glycopolymers. The neutral polymer is a block-type rhamnomannan partially should be substitutied by xylose residues, [→2)-α-[ß-D-Xylp-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼45. The acidic polymer has branched chain, bearing lactate and pyruvate residues, →4)-α-D-[S-Lac-(2-3)-α-L-Rhap-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-[4,6-R-Pyr]-D-Galp-(1 â†’ 3)-ß-D-Glcp-(1 â†’. The structures of both glycopolymers were not described in the Gram-positive bacteria to date. The glycopolymers were studied by chemical and NMR spectroscopic methods. The results of this study provide new data on diversity of bacterial glycopolymers and may prove useful in the taxonomy of the genus Rathayibacter and for understanding the molecular mechanisms of interaction between plants and plant endophytes.

Immune Response to Conjugates of Fragments of the Type K9 Capsular Polysaccharide of Acinetobacter baumannii with Carrier Proteins.

The clonal bacterial species Acinetobacter baumannii is an emerging multidrug-resistant pathogen which causes high-lethality infections. Cells of A. baumannii are surrounded by the type-specific capsular polysaccharide (CPS), which provides resistance to the protective mechanisms of the host and is considered a target for immunization. The conjugates of three inert carrier proteins and A. baumannii type K9 CPS fragments, which contained various numbers of oligosaccharide repeats (K-units), were synthesized by periodate oxidation and squaric acid chemistry. The conjugates were applied to immunize mice, and chemical synthesis by squaric acid was shown to significantly improve the immunogenic properties of glycoconjugate. In BALB/c mice, IgG antibodies were predominant among type K9 CPS reactive antibodies, and their total content was several times higher than that of IgM. Immune sera were characterized by their opsonization ability during practically the entire lives of the experimental mice. The sera were cross-reactive, but the highest specificity was observed against the antigen (type K9 CPS) used for immunization. The immunization of BALB/c and ICR-1 mice with a glycoconjugate without adjuvants led to varying degrees of stimulation of IL-10, IL-17A, and TNF-α production, but not IL-4 production in the ICR-1 mice. This is in contrast to the BALB/c mice, in which γ-IFN production was also activated. The protective effectiveness of the glycoconjugates obtained by squaric acid chemistry was demonstrated by experiments that involved challenging immunized and nonimmunized animals with a lethal dose of A. baumannii K9. IMPORTANCE Immunization by glycoconjugates with A. baumannii type K9 CPS fragments induced a high level of antibodies (predominantly IgG) in sera, which reacted specifically with the CPS of A. baumannii type K9, as well as a long immunological memory. The sera of immunized animals efficiently opsonized A. baumannii type K9. Immunization resulted in the balanced production of pro/anti-inflammatory lymphokines and protective antibodies to ensure the survival of the mice infected with A. baumannii. The level of specific antibodies was sufficient to provide protective immunity against the challenge by A. baumannii, making this approach applicable in the development of vaccine preparations.

Biosynthesis of the Pseudomonas aeruginosa common polysaccharide antigen by D-Rhamnosyltransferases WbpX and WbpY.

The Gram-negative bacterium Pseudomonas aeruginosa simultaneously expresses two O-antigenic glycoforms. While the O-specific antigen (OSA) is variable in composition, the common polysaccharide antigen (CPA) is highly conserved and is composed of a homopolymer of D-rhamnose (D-Rha) in trisaccharide repeating units [D-Rhaα1-2-D-Rhaα1-3-D-Rhaɑ1-3]n. We have previously reported that α3-D-Rha-transferase WbpZ transfers a D-Rha residue from GDP-D-Rha to D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl. Genes encoding two more D-Rha-transferases are found in the O antigen gene cluster (wbpX and wbpY). In this study we showed that WbpX and WbpY recombinantly expressed in E. coli differ in their donor and acceptor specificities and have properties of GT-B folded enzymes of the GT4 glycosyltransferase family. NMR spectroscopic analysis of the WbpY reaction product showed that WbpY transferred one D-Rha residue in α1-3 linkage to synthetic D-Rhaα1-3-D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl acceptor. WbpX synthesized several products that contained D-Rha in both α1-2 and α1-3 linkages. Mass spectrometry indicated that the mixture of WbpX and WbpY efficiently catalyzed the synthesis of D-Rha oligomers in a non-processive mechanism. Since O antigens are virulence factors, these findings open the door to advancing technology for antibacterial drug discovery and vaccine development.

The wclY gene of Escherichia coli serotype O117 encodes an α1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity.

The O antigen of enterotoxigenic Escherichia coli serotype O117 consists of repeating units with the structure [-D-GalNAcß1-3-L-Rhaα1-4-D-Glcα1-4-D-Galß1-3-D-GalNAcα1-4]n. A related structure is found in E. coli O107 where Glc is replaced by a GlcNAc residue. The O117 and O107 antigen biosynthesis gene clusters are homologous and reveal the presence of four putative glycosyltransferase (GT) genes, wclW, wclX, wclY and wclZ, but the enzymes have not yet been biochemically characterized. We show here that the His6-tagged WclY protein expressed in E. coli Lemo21(DE3) cells is an α1,4-Glc-transferase that transfers Glc to the Gal moiety of Galß1-3GalNAcα-OPO3-PO3-phenoxyundecyl as a specific acceptor and that the diphosphate moiety of this acceptor is required. WclY utilized UDP-Glc, TDP-Glc, ADP-Glc, as well as UDP-GlcNAc, UDP-Gal or UDP-GalNAc as donor substrates, suggesting an unusual broad donor specificity. Activity using GDP-Man suggested the presence of a novel Man-transferase in Lemo21(DE3) cells. Mutations of WclY revealed that both Glu residues of the Ex7E motif within the predicted GT domain are essential for activity. High GlcNAc-transferase (GlcNAc-T) activities of WclY were created by mutating Arg194 to Cys. A triple mutant identical to WclY in E. coli O107 was identified as an α1,4 GlcNAc-T. The characterization of WclY opens the door for the development of antibacterial approaches.

Group A, B, C, and G Streptococcus Lancefield antigen biosynthesis is initiated by a conserved α-d-GlcNAc-ß-1,4-l-rhamnosyltransferase.

Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-ß-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter-dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-ß-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance.

Synthesis of Phenoxyundecyl Diphosphate Disaccharides for Studies of the Biosynthesis of O Antigenic Polysaccharides in Enteric Bacteria.

The biosynthesis of O antigenic polysaccharides in enteric bacteria from nucleoside diphosphate sugars (donor substrates) is catalyzed by the corresponding glycosyltransferases and proceeds through the intermediate formation of undecaprenyl diphosphate sugars (acceptor substrates). To study this process, a chemical synthesis of the compounds having the natural structure or their modified analogs is necessary. The phosphoroimidazolidate method is a universal method for synthesis of lipid diphosphate disaccharides containing 2-acetamido-2-deoxyglycosyl residue at the reducing end of the disaccharide moiety and 11-phenoxyundecyl residue as lipid fragment of the molecule. We report here protocols to synthesize the disaccharides P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate [D-Rha(α1-3)-D-GlcNAcα-PP-PhU, Compound 1] and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate [D-Gal(ß1-3)-D-GalNAcα-PP-PhU, Compound 6]. We describe the procedures for identification and structure estimation of compounds by TLC, NMR, and MS. We also include the biochemical testing of Compound 6 with α2,3-sialyltransferase WbwA from Escherichia coli O104.

Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate for the investigation of biosynthesis of O-antigenic polysaccharides in Pseudomonas aeruginosa and Escherichia coli O104.

Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P1-(11-phenoxyundecyl)-P2- (2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively.

Identification and biochemical characterization of a novel α-1,3-mannosyltransferase WfcD from Escherichia coli O141.

Glycosyltransferases (GTs) catalyze the formation of regio- and stereospecific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the wfcD gene from the Escherichia coli O141 O-antigen gene cluster encoding an α-1,3-mannosyltransferase that catalyzed the formation of the linkage Man(α1-3)-GlcNAc was biochemically characterized. WfcD was expressed in E. coli BL21 (DE3), and the enzymatic product was identified by liquid chromatography-mass spectrometry (LC-MS), collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn) and glycosidase digestion using the donor substrate GDP-Man and the synthetic acceptor substrate decyl diphosphate 2-acetamido-2-deoxy-α-D-glucopyranose (GlcNAc-PP-De). The kinetic and physiochemical properties and the substrate specificity of WfcD were investigated. WfcD is the first characterized bacterial mannosyltransferase that acts on the Man(α1-3)-GlcNAc linkage. This study enhances our knowledge of the diverse functions of GTs.

Biochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3.

Glycosyltransferases (GTs) catalyze the formation of regio- and stereo-specific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the gene wclR from the Escherichia coli O3 O-antigen gene cluster that encodes an α 1, 3-galactosyltransferase (GalT) that acts on the linkage Gal α 1, 3-GlcNAc was biochemically characterized. WclR was expressed in E. coli BL21 (DE3), and the enzymatic product was identified by liquid chromatography-mass spectrometry (LC-MS), collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)) and galactosidase digestion, using UDP-Gal as the donor substrate and the synthetic acceptor substrate GlcNAc-PP-De (decyl diphosphate N-acetylglucosamine). The physiochemical properties and the substrate specificity of WclR were investigated. WclR is the first bacterial GalT characterized that acts on the linkage Gal α 1, 3-GlcNAc. This study enhanced our knowledge of the diversified functions of GTs and provided a novel enzyme source for possible pharmaceutical application.

Mass spectrometric characterization of a two-glycosyltransferase tandem reaction for assembly of tetrasaccharide repeating unit of Escherichia coli O77 O-antigen.

The wbaD gene and wbaC gene from Escherichia coli O77 O-antigen gene cluster encoding mannosyltransferases were functionally characterized in vitro. A synthetic acceptor P(1)-(11-phenoxyundecyl)-P(2)-(2-acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate (GlcNAc-PP-PhU) was used as an acceptor and GDP-Man as a donor substrate; the activities of WbaD and WbaC were confirmed by detailed structural characterization of their lipooligosacharide enzyme products using high-sensitivity negative-ion electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (CID) MS-MS. The extensive fragmentation unequivocally demonstrated that the Man(1-3)-GlcNAc linkage in WbaD catalyzed reaction product and two Man(1-2)-Man linkages in tandem WbaD/WbaC catalyzed reaction product are present, respectively. This study provided valuable information for the understanding of diversified glycosyltransferase (GT) functions and the two GTs characterized can serve as additional enzyme sources for possible pharmaceutical related applications.

The use of O-trifluoroacetyl protection and profound influence of the nature of glycosyl acceptor in benzyl-free arabinofuranosylation.

The influence of O-trifluoroacetyl (TFA) groups at different positions of thioglycoside glycosyl donors on stereoselectivity of α-arabinofuranosylation leading to corresponding disaccharides was studied. It was shown that TFA group in thioglycoside glycosyl donors, when combined with 2-O-(triisopropylsilyl) (TIPS) non-participating group, may be regarded as an electron-withdrawing protecting group that may enhance 1,2-cis-selectivity in arabinofuranosylation, the results strongly depending on the nature of glycosyl acceptor. The reactivities of the glycosyl donors were compared with those of a similar thioglycoside with O-pentafluoropropionyl groups and the known phenyl 3,5-O-(di-tert-butylsilylene)-1-thio-α-d-arabinofuranosides with 2-O-TIPS and 2-O-benzyl groups. The 'matching' in the donor-acceptor combination was found to be critical for achieving both high reactivity of glycosyl donor and ß-stereoselectivity of arabinofuranosylation. The use of glycosyl donors with TFA and silyl protection may be useful in the realization of the benzyl-free approach to oligoarabinofuranosides with azido group in aglycon-convenient building blocks for the preparation of neoglycoconjugates.

Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5.

The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P¹-[11-(anthracen-9-ylmethoxy)undecyl]-P²-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E. coli) O157 and for galactosyltransferase WbwC from E. coli O5. This is the first report of the Gal-transferase activity of the wbwC gene product of E.coli O5. The presence of the fluorescent label in the acceptor molecule allows the detection of glycosyltransferase reaction products with high sensitivity, eliminating the need for radioactive nucleotide sugars.

Synthesis of branched arabinofuranose pentasaccharide fragment of mycobacterial arabinans as 2-azidoethyl glycoside.

Branched arabinofuranose pentasaccharide with 2-azidoethyl aglycon was prepared for the first time by [3+1+1] bis-(1,2-cis)-glycosylation of trisaccharide diol with silyl-protected thioglycoside glycosyl donor. The presence of 2-azidoethyl aglycon would enable the preparation of neoglycoconjugates using the click chemistry approaches.

Biochemical characterization of WbdN, a ß1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157.

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcß1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was ß1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid ß1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.

Ring opening of acylated ß-d-arabinofuranose 1,2,5-orthobenzoates with nucleophiles allows access to novel selectively-protected arabinofuranose building blocks.

ß-d-Arabinofuranose 1,2,5-orthobenzoates with 3-O-acetyl, 3-O-benzoyl, and 3-O-chloroacetyl groups were prepared in an efficient manner starting from readily available crystalline methyl 2,3,5-tri-O-benzoyl-α-d-arabinofuranoside, and ring-opening reactions of these compounds with O- and S-nucleophiles were studied. Optimized conditions leading to the formation of the respective monosaccharide adducts (up to 96% isolated yields) and to α-(1→5)-linked disaccharide thioglycosides with 5'-OH unprotected (up to 30% isolated yields) were found. Basing on these results, a novel approach for effective differentiation of 3,5-diol system and 2-hydroxy group in arabinofuranose thioglycosides was proposed. The selectively protected derivatives prepared are valuable building blocks for the assembly of linear and branched oligoarabinofuranosides.

11-Phenoxyundecyl phosphate as a 2-acetamido-2-deoxy-α-d-glucopyranosyl phosphate acceptor in O-antigen repeating unit assembly of Salmonella arizonae O:59.

A synthesis of 11-phenoxyundecyl phosphate and its biochemical transformation (using GlcNAc-P transferase from Salmonella arizonae O:59 membranes catalysing transfer of GlcNc-phosphate from UDP-GlcNAc on lipid-phosphate) into P(1)-11-phenoxyundecyl, P(2)-2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate are described.

Use of methanesulfonic acid in the reductive ring-opening of O-benzylidene acetals.

Methanesulfonic acid was shown to be an efficient and convenient substitute for ethereal HCl in reductive 4,6-O-benzylidene acetal ring-opening reaction with sodium cyanoborohydride in THF. Normal regioselectivity was observed, the 6-O-benzyl ethers with free 4-OH group being the major products of the reaction.

Structure of an acidic polysaccharide from the agar-decomposing marine bacterium Pseudoalteromonas atlantica strain IAM 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid.

The structure of an acidic polysaccharide from Pseudoalteromonas atlantica strain 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac) has been elucidated. The polysaccharide was studied by 1H and 13C NMR spectroscopy, including 2D experiments, along with sugar and methylation analyses. After a selective hydrolysis a modified polysaccharide devoid of its side chain could be isolated. It was found that the polysaccharide has pentasaccharide repeating units with following structure: [structure: see text].

Structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6.

The structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6 has been elucidated. Chemical modifications of the polymer in conjunction with 1H and 13C NMR spectroscopy, including 2D techniques, were employed in the analysis. It is concluded that the repeating unit is composed of two nine-carbon sugars as follows: -->4)-alpha-NonpA-(2-->3)-beta-Sugp-(1--> where alpha-NonpA is 5-acetamido-7-acetamidino-8-O-acetyl-3,5,7,9-tetradeoxy-L-glycero-alpha-D-galacto-non-2-ulosonic acid (8eLeg) and beta-Sugp is 2-acetamido-2,6-dideoxy-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-beta-D-galactopyranose, with the proposed name Shewanellose (She).