The antigen-binding site of an N-propionylated polysialic acid-specific antibody protective against group B meningococci is consistent with extended epitopes.

Monoclonal antibodies 13D9 and 6B9 are both specific for N-propionylated polysialic acid (NPrPSA); however, while 13D9 is protective against meningitis caused by group B meningococci and Escherichia coli capsular type K1 infection, 6B9 is not. The crystal structures of the Fabs from the two antibodies determined at 2.06 and 2.45 Å resolutions, respectively, reveal markedly different combining sites, where only the surface of 13D9 is consistent with the recognition of extended helical epitopes known to exist in the capsular polysaccharides of etiological agents of meningitis. Interestingly, complementarity determining region H2 on 13D9 lies in a non-canonical conformation that docking studies show is a critical feature in the generation of negative free energy of binding. Finally, the model of extended NPrPSA decasaccharide bound to 13D9 derived from docking studies is consistent with saturation transfer difference nuclear magnetic resonance experiments. Together, these results provide further evidence that extended epitopes have the ability to break immune tolerance associated with the polysialic acid capsule of these pathogens.

Cell surface glycoproteins from Thermoplasma acidophilum are modified with an N-linked glycan containing 6-C-sulfofucose.

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34-48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a "general" protein N-glycosylation system that targets a number of cell surface proteins.

The CMP-legionaminic acid pathway in Campylobacter: biosynthesis involving novel GDP-linked precursors.

The sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid, or legion-aminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an alpha2,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.

Structural elucidation of the novel core oligosaccharide from LPS of Burkholderia cepacia serogroup O4.

Lipopolysaccharide (LPS) is an important virulence factor of Burkholderia cepacia, an opportunistic bacterial pathogen that causes life-threatening disease in cystic fibrosis patients and immunocompromised individuals. B. cepacia LPS comprises an O-specific polysaccharide covalently linked to a core oligosaccharide (OS) which in turn is attached to a lipid A moiety. The complete structure of the LPS core oligosaccharide from B. cepacia serotype O4 was investigated by detailed NMR and mass spectrometry (MS) methods. High- (HMW) and low-molecular-weight (LMW) OSs were obtained by deacylation, dephosphorylation, and reducing-end reduction of the LPS. Glycan and NMR analyses established that both OSs contain a common inner-core structure consisting of D-glucose, L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, 3-deoxy-D-manno-octulsonic acid, and D-glycero-D-talo-2-octulosonic acid. The structure of the LMW OS differed from that of the HMW OS in that it lacks a tetra-rhamnosyl GlcNAc OS extension. These structural conclusions were confirmed by tandem MS analyses of the two OS fractions as well as an OS fraction obtained by alkaline deacylation of the LPS. The location of a phosphoethanolamine substituent in the core region was determined by ESI-MS and methylation analysis of O-deacylated LPS and core OS samples. A polyclonal antibody to B. cepacia serotype O4 core OS was cross-reactive with several other serotypes indicating common structural features.

Flagellar glycosylation in Clostridium botulinum.

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens.

The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, beta-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-beta-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-alpha-D-GlcNAc (UDP-N-acetyl-alpha-D-glucosamine) is converted via four steps into UDP-alpha-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-alpha-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-mannuronic acid). UDP-alpha-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6-WbpI and His6-WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-alpha-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-alpha-D-GlcNAc, UDP-alpha-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-alpha-D-glucuronic acid) or UDP-alpha-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-alpha-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme-substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6-WbpI catalysed the 2-epimerization of UDP-alpha-D-GlcNAc3NAcA, converting it into UDP-alpha-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases.

The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group.

A recent study that examined multiple strains of Campylobacter jejuni reported that HS:19, a serostrain that has been associated with the onset of Guillain-Barré syndrome, had unidentified labile, capsular polysaccharide (CPS) structures. In this study, we expand on this observation by using current glyco-analytical technologies to characterize these unknown groups. Capillary electrophoresis electrospray ionization MS and NMR analysis with a cryogenically cooled probe (cold probe) of CPS purified using a gentle enzymatic method revealed a hyaluronic acid-type [-4)-beta-D-GlcA6NGro-(1-3)-beta-D-GlcNAc-(1-]n repeating unit, where NGro is 2-aminoglycerol. A labile alpha-sorbofuranose branch located at C2 of GlcA was determined to have the L configuration using a novel pyranose oxidase assay and is the first report of this sugar in a bacterial glycan. A labile O-methyl phosphoramidate group, CH3OP(O)(NH2)(OR) (MeOPN), was found at C4 of GlcNAc. Structural heterogeneity of the CPS was due to nonstoichiometric glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, variably methylated phosphoramidate group. Examination of whole bacterial cells using high-resolution magic angle spinning NMR revealed that the MeOPN group is a prominent feature on the cell surface for this serostrain. These results are reminiscent of those in the 11168 and HS:1 strains and suggest that decoration of CPS with nonstoichiometric elements such as keto sugars and the phosphoramidate is a common mechanism used by this bacterium to produce a structurally complex surface glycan from a limited number of genes. The findings of this work with the HS:19 serostrain now present a means to explore the role of CPS as a virulence factor in C. jejuni.

The HS:1 serostrain of Campylobacter jejuni has a complex teichoic acid-like capsular polysaccharide with nonstoichiometric fructofuranose branches and O-methyl phosphoramidate groups.

Recently, the CPS biosynthetic loci for several strains of Campylobacter jejuni were sequenced and revealed evidence for multiple mechanisms of structural variation. In this study, the CPS structure for the HS:1 serostrain of C. jejuni was determined using mass spectrometry and NMR at 600 MHz equipped with an ultra-sensitive cryogenically cooled probe. Analysis of CPS purified using a mild enzymatic method revealed a teichoic acid-like [-4)-alpha-d-Galp-(1-2)-(R)-Gro-(1-P](n), repeating unit, where Gro is glycerol. Two branches at C-2 and C-3 of galactose were identified as beta-d-fructofuranoses substituted at C-3 with CH(3)OP(O)(NH(2))(OR) groups. Structural heterogeneity was due to nonstoichiometric glycosylation at C-3 of galactose and variable phosphoramidate groups. Identical structural features were found for cell-bound CPS on intact cells using proton homonuclear and (31)P heteronuclear two-dimensional HR-MAS NMR at 500 MHz. In contrast, spectroscopic data acquired for hot water/phenol purified CPS was complicated by the hydrolysis and subsequent loss of labile groups during extraction. Collectively, the results of this study established the importance of using sensitive isolation techniques and HR-MAS NMR to examine CPS structures in vivo when labile groups are present. This study uncovered how incorporation of variable O-methyl phosphoramidate groups on nonstoichiometric fructose branches is used in C. jejuni HS:1 as a strategy to produce a highly complex polysaccharide from its small CPS biosynthetic locus and a limited number of sugars.

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents.

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.

Development of specific inhibitors for heparin-binding proteins based on the cobra cardiotoxin structure: an effective synthetic strategy for rationally modified heparin-like disaccharides and a trisaccharide.

Recently, a new heparin disaccharide-binding site on the convex side of cobra cardiotoxin (CTX) was identified by NMR spectroscopy and molecular modeling. To further characterize this site two heparin-like disaccharides were synthesized for binding studies with CTX, and a trisaccharide was synthesized for testing the sequence of the disaccharide binding to CTX. Thus six differentially protected monosaccharide building blocks (three l-iduronic acids and three d-glucosamines) were prepared. These include a l-iduronic acid elongation building block namely methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-alpha-l-idopyranosyluronate trichloroacetimidate for which a single-crystal X-ray structure was determined to have M(r)=576.79, a=9.3098(11)A alpha=90 degrees , b=10.3967(12)A beta=90 degrees , c=28.026(3)A gamma=90 degrees , V=2712.7(6)A(3), P2(1)2(1)2(1), Z=4, mu=0.71073A, and R=0.0378 for 7586 observed reflections. It shows that the molecular structure of the donor is in the (1)C(4) conformation with significant 1,3-diaxial interactions between O-1 and O-3 as well as O-2 and O-4. The disaccharides and trisaccharide vary in the degree and position of O- and N-sulfation. The pivaloyl group was used as permanent protecting group of hydroxyl. The levulinoyl group was used as the temporary protecting group to protect the hydroxyl for elongation.

Structural characterization of the O-chain polysaccharide isolated from Bordetella avium ATCC 5086: variation on a theme(1).

The O-chain polysaccharide (O-PS) of Bordetella avium was isolated from the lipopolysaccharide by mild acid hydrolysis to remove the lipid A, followed by hydrofluorolysis to remove the lipopolysaccharide core oligosaccharide leaving a residual O-PS for structural analysis. High resolution (1)H and (13)C NMR and MALDI studies showed the O-chain to be a polymer composed of 1,4-linked 2-acetamidino-3-[3-hydroxybutanamido]-2,3-dideoxy-beta-D-glucopyranosyluronic acid residues.

Structural analysis of the lipopolysaccharide from Neisseria meningitidis strain BZ157 galE: localisation of two phosphoethanolamine residues in the inner core oligosaccharide.

The structure of the phase-variable lipopolysaccharide (LPS) from the group B Neisseria meningitidis strain BZ157 galE was elucidated. The structural basis for the LPS's variation in reactivity with a monoclonal antibody (MAb) B5 that has specificity for the presence of phosphoethanolamine (PEtn) at the 3-position of the distal heptose residue (HepII) was established. The structure of the O-deacylated LPS was deduced by a combination of monosaccharide analyses, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These analyses revealed the presence of a novel inner core oligosaccharide (OS) structure in the MAb B5 reactive (B5+) LPS that contained two PEtn residues simultaneously substituting the 3- and 6-positions of the HepII residue. The determination of this structure has identified a further degree of variability within the inner core OS of meningococcal LPS that could contribute to the interaction of meningococcal strains with their host.

Structural analysis of the lipopolysaccharide derived core oligosaccharides of Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and the genome strain 5b.

The structures of the core oligosaccharides of the lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and 5b were elucidated. The LPS's were subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the core oligosaccharides were determined on the basis of the combined data from these experiments. [carbohydrate formula see text] For serotype 1: R is (1S)-GalaNAc-(1-->4,6)-alpha-Gal II-(1-->3)-beta-Gal I-(1-->, and R' is H For serotype 2: R is beta-Glc III-(1-->, and R' is D-alpha-D-Hep V-(1--> For serotypes 5a and 5b: R is H and R' is D-alpha-D-Hep V-(1--> All oligosaccharides elaborated a conserved inner core structure, as illustrated. All sugars were in the pyranose ring form apart from the open-chain N-acetylgalactosamine, the identification of which in the serotype 1 LPS was of interest.

Comparison of lipopolysaccharide structures of Bordetella pertussis clinical isolates from pre- and post-vaccine era.

Endotoxins are lipopolysaccharides (LPS), and major constituents of the outer membrane of Gram-negative bacteria. Bordetella pertussis LPS were the only major antigens, of this agent of whooping-cough, that were not yet analyzed on isolates from the pre- and post-vaccination era. We compared here the LPS structures of four clinical isolates with that of the vaccine strain BP 1414. All physico-chemical analyses, including SDS-PAGE, TLC, and different MALDI mass spectrometry approaches were convergent. They helped demonstrating that, on the contrary to some other B. pertussis major antigens, no modification occurred in the dodecasaccharide core structure, as well as in the whole LPS molecules. These results are rendering these major antigens good potential vaccine components. Molecular modeling of this conserved LPS structure also confirmed the conclusions of previous experiments leading to the production of anti-LPS monoclonal antibodies and defining the main epitopes of these major antigens.

Structural characterization of surface glycans from Clostridium difficile.

Whole-cell high-resolution magic angle spinning (HR-MAS) NMR was employed to survey the surface polysaccharides of a group of clinical and environmental isolates of Clostridium difficile. Results indicated that a highly conserved surface polysaccharide profile among all strains studied. Multiple additional peaks in the anomeric region were also observed which prompted further investigation. Structural characterization of the isolated surface polysaccharides from two strains confirmed the presence of the conserved water soluble polysaccharide originally described by Ganeshapillai et al. which was composed of a hexaglycosyl phosphate repeat consisting of [→6)-ß-D-Glcp-(1-3)-ß-D-GalpNAc-(1-4)-α-D-Glcp-(1-4)-[ß-D-Glcp(1-3]-ß-D-GalpNAc-(1-3)-α-D-Manp-(1-P→]. In addition, analysis of phenol soluble polysaccharides revealed a similarly conserved lipoteichoic acid (LTA) which could be detected on whole cells by HR-MAS NMR. Conventional NMR and mass spectrometry analysis indicated that the structure of this LTA consisted of the repeat unit [→6)-α-D-GlcpNAc-(1-3)-[→P-6]-α-D-GlcpNAc-(1-2)-D-GroA] where GroA is glyceric acid. The repeating units were linked by a phosphodiester bridge between C-6 of the two GlcNAc residues (6-P-6). A minor component consisted of GlcpN-(1-3) instead of GlcpNAc-(1-3) in the repeat unit. Through a 6-6 phosphodiester bridge this polymer was linked to →6)-ß-D-Glcp-(1-6)-ß-D-Glcp-(1-6)-ß-D-Glcp-(1-1)-Gro, with glycerol (Gro) substituted by fatty acids. This is the first report of the utility of HR-MAS NMR in the examination of surface carbohydrates of Gram positive bacteria and identification of a novel LTA structure from Clostridium difficile.

The application of NMR spectroscopy to functional glycomics.

Glycomics which is the study of saccharides and genes responsible for their formation requires the continuous development of rapid and sensitive methods for the identification of glycan structures. It involves glycoanalysis which relies upon the development of methods for determining the structure and interactions of carbohydrates. For the application of functional glycomics to microbial virulence, carbohydrates and their associated metabolic and carbohydrate processing enzymes and respective genes can be identified and exploited as targets for drug discovery, glyco-engineering, vaccine design, and detection and diagnosis of diseases. Glycomics also encompasses the detailed understanding of carbohydrate-protein interactions and this knowledge can be applied to research efforts focused toward the development of vaccines and immunological therapies to alleviate infectious diseases.

The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-D-rhamnose biosynthesis pathway.

The rare 6-deoxysugar D-rhamnose is a component of bacterial cell surface glycans, including the D-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-D-rhamnose synthesis from GDP-D-mannose is catalyzed by two enzymes. The first is a GDP-D-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-D-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-D-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-D-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-D-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 A resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.

Flagellin glycosylation in Pseudomonas aeruginosa PAK requires the O-antigen biosynthesis enzyme WbpO.

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

Recognition characteristics of monoclonal antibodies that are cross-reactive with gangliosides and lipooligosaccharide from Campylobacter jejuni strains associated with Guillain-Barré and Fisher syndromes.

The enteropathogen Campylobacter jejuni has the ability to synthesize glycan structures that are similar to mammalian gangliosides within the core component of its lipooligosaccharide (LOS). Exposure to ganglioside mimics in some individuals results in the production of autoantibodies that deleteriously attack nerve surface gangliosides, precipitating the onset of Guillain-Barré and Fisher syndromes (GBS and FS). We have characterized the interaction of four monoclonal antibodies (mAbs), established by sensitization of mice with LOS isolated from GBS- and FS-associated C. jejuni strains, with chemoenzymatically synthesized gangliooligosaccharides. Surface plasmon resonance (SPR) measurements demonstrate that three of the mAbs interact specifically with derivatives corresponding to their targeted gangliosides, with dissociation constants ranging from 10 to 20 microM. Antibody binding to the gangliooligosaccharides was probed by saturation transfer difference (STD) NMR spectroscopy. STD signals, resulting from antibody/oligosaccharide interaction, were observed for each of the four mAbs. In two cases, differential saturation transfer rates to oligosaccharide resonances enabled detailed epitope mapping. The binding of GD1a-S-Phe with GB1 is characterized by close association of the immunoglobulin with sites that are distributed over several residues of the oligosaccharide. This contrasts sharply with the profile observed for the binding of both GD3-S-Phe and GT1a-S-Phe with FS1. The close antigenic contacts in these ganglioside derivatives are confined to the N-acetylmannosaminyl portion of the terminal N-acetylneuraminic acid (NeuAc) residue of the disialosyl moiety. Our characterization of FS1 provides insight, at an atomic level, into how a single antigenic determinant presented by the LOS of C. jejuni can give rise to antibodies with binding promiscuity to [alphaNeuAc-(2-8)-alphaNeuAc]-bound epitopes and demonstrates why sera from FS patients have antibodies that are often reactive with more than one disialylated ganglioside.

Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with mycobacterium-like alpha-1,5-linked d-Araf oligosaccharides.

Pseudomonas aeruginosa is a gram-negative bacterium that uses polar type IV pili for adherence to various materials and for rapid colonization of surfaces via twitching motility. Within the P. aeruginosa species, five distinct alleles encoding variants of the structural subunit PilA varying in amino acid sequence, length, and presence of posttranslational modifications have been identified. In this work, a combination of mass spectrometry and nuclear magnetic resonance spectroscopy was used to identify a novel glycan modification on the pilins of the group IV strain Pa5196. Group IV pilins continued to be modified in a lipopolysaccharide (wbpM) mutant of Pa5196, showing that, unlike group I strains, the pilins of group IV are not modified with the O-antigen unit of the background strain. Instead, the pilin glycan was determined to be an unusual homo-oligomer of alpha-1,5-linked d-arabinofuranose (d-Araf). This sugar is uncommon in prokaryotes, occurring mainly in the cell wall arabinogalactan and lipoarabinomannan (LAM) polymers of mycobacteria, including Mycobacterium tuberculosis and Mycobacterium leprae. Antibodies raised against M. tuberculosis LAM specifically identified the glycosylated pilins from Pa5196, confirming that the glycan is antigenically, as well as chemically, identical to those of Mycobacterium. P. aeruginosa Pa5196, a rapidly growing strain of low virulence that expresses large amounts of glycosylated type IV pilins on its surface, represents a genetically tractable model system for elucidation of alternate pathways for biosynthesis of d-Araf and its polymerization into mycobacterium-like alpha-1,5-linked oligosaccharides.