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1.
Glycobiology ; 23(8): 946-54, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23704298

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/chemistry , Antigens, Bacterial/chemistry , Binding Sites, Antibody , Epitopes/chemistry , Polysaccharides, Bacterial/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Epitopes/immunology , Epitopes/metabolism , Molecular Docking Simulation , Neisseria meningitidis/chemistry , Neisseria meningitidis/immunology , Polysaccharides, Bacterial/immunology , Polysaccharides, Bacterial/metabolism
2.
Carbohydr Res ; 378: 45-55, 2013 Aug 30.
Article in English | MEDLINE | ID: mdl-23374752

ABSTRACT

In order to evaluate the importance of molecular shape of inhibitor molecules and the charge/H-bond and hydrophobic interactions, we synthesized three types of molecules and tested them against a sialyltransferase. The first type of compounds were designed as substrate mimics in which the phosphate in CMP-Neu5NAc was replaced by a non-hydrolysable, uncharged 1,2,3-triazole moiety. The second type of compound contained a 2-deoxy-2,3-dehydro-acetylneuraminic moiety which was linked to cytidine through its carboxylic acid and amide linkers. In the third type of compound the sialyl phosphate was substituted by an aryl sulfonamide which was then linked to cytidine. Inhibition study of these cytidine conjugates against Campylobacter jejuni sialyltransferase Cst 06 showed that the first type of molecules are competitive inhibitors, whereas the other two could only inhibit the enzyme non-competitively. The results indicate that although the binding specificity may be guided by molecular shape and H-bond interaction, the charge and hydrophobic interactions contributed most to the binding affinity.


Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydrophobic and Hydrophilic Interactions , Sialyltransferases/antagonists & inhibitors , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Campylobacter jejuni/enzymology , Enzyme Inhibitors/chemical synthesis
3.
J Biomed Mater Res A ; 101(3): 787-96, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22961935

ABSTRACT

Photocaged RGDS is a cell nonadhesive tetrapeptide that can be activated with light to become cell-adhesive. Such molecules can find useful applications in controlling cell adhesion for biological study, drug development, and in forming dynamic, adhesion-controlled biomaterials. Herein, we prepared RGDS peptide photocaged either on the Arg-Gly backbone amide nitrogen atom (R[-]GDS) or Asp side chain carboxyl (RG[D]S). A critical comparison of the peptides' chemical and physiological properties relevant for biological applications was carried out. It was observed that RG[D]S was synthesized more readily via automated solid-phase synthesis, underwent uncaging with a rate constant 3-fold higher than R[-]GDS, and was more stable in aqueous solution. Automated docking studies were performed to examine the interactions of various caged RGDS peptides with cell surface integrin receptor to identify suitable locations for the photosensitive 2-nitrobenzyl (NB) group for biological applications. A competitive binding ELISA method compared the ability of various peptides to bind to α(V)ß(3) cell integrin receptors and the data were found to be consistent with the modeling predictions. Finally, the application of our caged RGDS peptides in controlling cell adhesion to form cell patterns on a hydrogel material was presented.


Subject(s)
Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/chemical synthesis , Integrin alphaVbeta3/chemistry , Oligopeptides/chemistry , Oligopeptides/chemical synthesis , Animals , Cell Adhesion , Humans , Integrin alphaVbeta3/metabolism , Molecular Docking Simulation , Photochemical Processes , Protein Binding
4.
J Mol Biol ; 425(1): 186-97, 2013 Jan 09.
Article in English | MEDLINE | ID: mdl-23103756

ABSTRACT

Increasingly, experimental data on biological systems are obtained from several sources and computational approaches are required to integrate this information and derive models for the function of the system. Here, we demonstrate the power of a logic-based machine learning approach to propose hypotheses for gene function integrating information from two diverse experimental approaches. Specifically, we use inductive logic programming that automatically proposes hypotheses explaining the empirical data with respect to logically encoded background knowledge. We study the capsular polysaccharide biosynthetic pathway of the major human gastrointestinal pathogen Campylobacter jejuni. We consider several key steps in the formation of capsular polysaccharide consisting of 15 genes of which 8 have assigned function, and we explore the extent to which functions can be hypothesised for the remaining 7. Two sources of experimental data provide the information for learning-the results of knockout experiments on the genes involved in capsule formation and the absence/presence of capsule genes in a multitude of strains of different serotypes. The machine learning uses the pathway structure as background knowledge. We propose assignments of specific genes to five previously unassigned reaction steps. For four of these steps, there was an unambiguous optimal assignment of gene to reaction, and to the fifth, there were three candidate genes. Several of these assignments were consistent with additional experimental results. We therefore show that the logic-based methodology provides a robust strategy to integrate results from different experimental approaches and propose hypotheses for the behaviour of a biological system.


Subject(s)
Artificial Intelligence , Campylobacter jejuni/metabolism , Logic , Models, Biological , Polysaccharides, Bacterial/genetics , Systems Biology/methods , Bacterial Capsules/genetics , Bacterial Capsules/metabolism , Biosynthetic Pathways/genetics , Campylobacter jejuni/genetics , Gene Knockout Techniques , Genes, Bacterial/genetics , Genes, Bacterial/physiology , Glycomics , Metabolomics , Molecular Sequence Annotation , Mutation , Oligonucleotide Array Sequence Analysis , Phenotype , Polysaccharides, Bacterial/metabolism
5.
Carbohydr Res ; 354: 65-73, 2012 Jun 01.
Article in English | MEDLINE | ID: mdl-22560631

ABSTRACT

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.


Subject(s)
Clostridioides difficile/chemistry , Polysaccharides/chemistry , Carbohydrate Conformation , Clostridioides difficile/cytology , Magnetic Resonance Spectroscopy , Models, Molecular , Polysaccharides/isolation & purification , Surface Properties
6.
J Biol Chem ; 286(14): 12361-70, 2011 Apr 08.
Article in English | MEDLINE | ID: mdl-21257763

ABSTRACT

Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering Guillain-Barré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumor-associated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and Pk antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity.


Subject(s)
Campylobacter jejuni/metabolism , Gangliosides/metabolism , Lipopolysaccharides/metabolism , Bacterial Proteins/metabolism , Galactosyltransferases/metabolism , Lipopolysaccharides/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Sialyltransferases/metabolism , Substrate Specificity
7.
Methods Mol Biol ; 600: 155-73, 2010.
Article in English | MEDLINE | ID: mdl-19882127

ABSTRACT

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.


Subject(s)
Glycomics/methods , Magnetic Resonance Spectroscopy/methods , Biochemistry/methods , Carbohydrates/chemistry , Endopeptidase K/chemistry , Glycopeptides/chemistry , Glycoproteins/chemistry , Humans , Ligands , Models, Chemical , Molecular Biology/methods , Polysaccharides/chemistry , Protein Binding , Software
8.
Glycobiology ; 19(2): 153-9, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18955372

ABSTRACT

We have identified an alpha1,4-galactosyltransferase (CgtD) and a beta1,3-N-acetylgalactosaminyltransferase (CgtE) in the lipooligosaccharide (LOS) locus of Campylobacter jejuni LIO87. Strains that carry these genes may have the capability of synthesizing mimics of the P blood group antigens of the globoseries glycolipids. We have also identified an alpha1,3-N-acetylgalactosaminyltransferase (Pm1138) from Pasteurella multocida Pm70, which is involved in the synthesis of an LOS-bound Forssman antigen mimic and represents the only known bacterial glycosyltransferase with this specificity. The genes encoding the three enzymes were cloned and expressed in Escherichia coli as soluble recombinant proteins that can be used to chemoenzymatically synthesize the Forssman antigen, and its biosynthetic precursors, in high yields.


Subject(s)
Campylobacter jejuni/enzymology , Forssman Antigen/biosynthesis , Forssman Antigen/chemistry , Glycosyltransferases/chemistry , Pasteurella multocida/enzymology , Campylobacter jejuni/metabolism , Cloning, Molecular , Glycosyltransferases/metabolism , Magnetic Resonance Spectroscopy , Pasteurella multocida/metabolism
9.
Biochemistry ; 48(2): 220-2, 2009 Jan 20.
Article in English | MEDLINE | ID: mdl-19105626

ABSTRACT

High-resolution binding profiles were elucidated for anti-GM1 IgM autoantibodies from two patients with a progressive form of paraproteinemic polyneuropathy. Antibody-ligand interaction was characterized by generating STD-NMR signals in target ganglio-oligosaccharides added directly to patient sera, without the requirement of antibody fractionation. Both immunoglobulins were found to have similar binding modalities, with interaction confined to two distinct spatially separated regions of GM1: the terminal betaGal(1-3)betaGalNAc disaccharide unit and the sialic acid residue. We describe a unique and powerful biophysical technique applied to define the molecular interaction between autoimmune disease-causing antibodies and their ganglioside targets.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , Autoantibodies/blood , Biophysical Phenomena , Gangliosides/immunology , Nuclear Magnetic Resonance, Biomolecular/methods , Antibodies/immunology , Autoantibodies/immunology , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Epitopes , Humans , Ligands , Models, Molecular , Polyneuropathies/diagnosis , Polyneuropathies/immunology , Sensitivity and Specificity
10.
Glycobiology ; 19(3): 267-76, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19029107

ABSTRACT

A difficulty in explaining the mechanism whereby archaeal lipid membrane vesicles (archaeosomes) deliver entrapped protein antigens to the MHC class I cytosolic pathway from phagolysosomes of antigen-presenting cells has been the observation that they tend not to fuse. Here, we determine that archaeosomes, composed of archaeal isoprenoid mixtures of glyco and phospholipids, can be highly fusogenic when exposed to the pH and enzymes found in late phagolysosomes. Fusions were strictly dependent on acidic pH and the presence of alpha- or beta-glucosidase. Resonance energy transfer (RET) assays demonstrated that fusion conditions induced lipid mixing of archaeosome lipids with self-unlabeled archaeosomes. Because PC/PG/cholesterol liposomes by themselves did not fuse, it was possible to unequivocally show a fusion of rhodamine-labeled liposomes with archaeosomes by fluorescence microscopy and to demonstrate lipid mixing between labeled liposomes and archaeosomes by the RET assay. Radiotracer and (1)H NMR studies revealed that glycolipids in fused archaeosomes were not degraded significantly by glucosidase treatment during fusion. Rather, the glucosidases dramatically induced small archaeosomes to rapidly and visually aggregate at pH 4.8, but not 6.8, thus bringing membranes together appropriately as a first step in the fusion process. (1)H NMR was used to demonstrate that conditions causing aggregation correlated with binding of glucosidase to the archaeosomes. Binding at acidic pH occurred by the electrostatic interaction of positively charged glucosidase with the anionic phospholipids, although the interaction also occurred with the gentiobiosyl lipids. The data indicate a mechanism of membrane-membrane fusion for archaeal glycolipid membranes induced by glycosidase and illustrate the importance for inclusion of glycolipids in compositions of vesicles designed to deliver protein antigens to the cytosol for MHC class I presentation.


Subject(s)
Acids , Glycoside Hydrolases/pharmacology , Liposomes/immunology , Membrane Fusion/immunology , Terpenes/chemistry , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/metabolism , Glycolipids/chemistry , Glycolipids/immunology , Hydrogen-Ion Concentration , Light , Liposomes/chemistry , Liposomes/metabolism , Membranes/immunology , Membranes/metabolism , Nuclear Magnetic Resonance, Biomolecular , Phagosomes/chemistry , Phagosomes/metabolism , Phospholipases/pharmacology , Phospholipids/chemistry , Phospholipids/immunology , Rhodamines/metabolism , Scattering, Radiation , Static Electricity , Temperature , Time Factors
11.
FEBS J ; 275(17): 4428-44, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18671733

ABSTRACT

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.


Subject(s)
Clostridium botulinum/metabolism , Flagella/metabolism , Flagellin/metabolism , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Clostridium botulinum/genetics , Electrophoresis, Polyacrylamide Gel , Flagellin/chemistry , Genome, Bacterial , Glycosylation , Mice , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Tandem Mass Spectrometry
12.
Biophys Chem ; 132(1): 47-54, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17976897

ABSTRACT

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, (2)H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S(mol)) and dynamics (T(1)) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a "transient site", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel.


Subject(s)
Anesthetics, Local/chemistry , Etidocaine/chemistry , Lecithins/chemistry , Lidocaine/chemistry , Lipid Bilayers/chemistry , Animals , Electron Spin Resonance Spectroscopy , Fluorescence , Fluorescent Dyes/analysis , Liposomes , Magnetic Resonance Spectroscopy , Spin Labels
13.
J Bacteriol ; 190(3): 1097-107, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17993532

ABSTRACT

The enteric pathogen Campylobacter jejuni is a highly prevalent yet fastidious bacterium. Biofilms and surface polysaccharides participate in stress survival, transmission, and virulence in C. jejuni; thus, the identification and characterization of novel genes involved in each process have important implications for pathogenesis. We found that C. jejuni reacts with calcofluor white (CFW), indicating the presence of surface polysaccharides harboring beta1-3 and/or beta1-4 linkages. CFW reactivity increased with extended growth, under 42 degrees C anaerobic conditions, and in a DeltaspoT mutant defective for the stringent response (SR). Conversely, two newly isolated dim mutants exhibited diminished CFW reactivity as well as growth and serum sensitivity differences from the wild type. Genetic, biochemical, and nuclear magnetic resonance analyses suggested that differences in CFW reactivity between wild-type and DeltaspoT and dim mutant strains were independent of well-characterized lipooligosaccharides, capsular polysaccharides, and N-linked polysaccharides. Targeted deletion of carB downstream of the dim13 mutation also resulted in CFW hyporeactivity, implicating a possible role for carbamoylphosphate synthase in the biosynthesis of this polysaccharide. Correlations between biofilm formation and production of the CFW-reactive polymer were demonstrated by crystal violet staining, scanning electron microscopy, and confocal microscopy, with the C. jejuni DeltaspoT mutant being the first SR mutant in any bacterial species identified as up-regulating biofilms. Together, these results provide new insight into genes and processes important for biofilm formation and polysaccharide production in C. jejuni.


Subject(s)
Benzenesulfonates/metabolism , Biofilms/growth & development , Campylobacter jejuni/growth & development , Fluorescent Dyes/metabolism , Heat-Shock Response , Polysaccharides, Bacterial/metabolism , Up-Regulation , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Campylobacter jejuni/genetics , Campylobacter jejuni/metabolism , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Humans , Mutation , Polysaccharides, Bacterial/chemistry , Pyrophosphatases/genetics , Pyrophosphatases/metabolism
15.
J Biol Chem ; 282(39): 28566-28576, 2007 Sep 28.
Article in English | MEDLINE | ID: mdl-17675288

ABSTRACT

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.


Subject(s)
Amides/metabolism , Bacterial Capsules/metabolism , Campylobacter jejuni/metabolism , Phosphoric Acids/metabolism , Polysaccharides, Bacterial/metabolism , Animals , Bacterial Capsules/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bird Diseases/diagnosis , Bird Diseases/genetics , Bird Diseases/metabolism , Bird Diseases/microbiology , Bird Diseases/therapy , Campylobacter Infections/diagnosis , Campylobacter Infections/genetics , Campylobacter Infections/metabolism , Campylobacter Infections/microbiology , Campylobacter Infections/therapy , Campylobacter jejuni/genetics , Cecum/metabolism , Cecum/microbiology , Chickens , Enteritis/diagnosis , Enteritis/genetics , Enteritis/metabolism , Enteritis/microbiology , Enteritis/therapy , Genetic Complementation Test , Humans , Magnetic Resonance Spectroscopy , Miller Fisher Syndrome/diagnosis , Miller Fisher Syndrome/genetics , Miller Fisher Syndrome/metabolism , Miller Fisher Syndrome/microbiology , Miller Fisher Syndrome/therapy , Multigene Family/genetics , Mutation , Polysaccharides, Bacterial/genetics , Transferases/genetics , Transferases/metabolism , Typhlitis/diagnosis , Typhlitis/genetics , Typhlitis/metabolism , Typhlitis/microbiology , Typhlitis/therapy
16.
Biochemistry ; 46(27): 8164-71, 2007 Jul 10.
Article in English | MEDLINE | ID: mdl-17567050

ABSTRACT

The non-typeable Haemophilus influenzae strain DH1 was isolated from a 25 year old male patient with Fisher syndrome, a postinfectious autoimmune condition characterized by the presence of anti-GQ1b IgG antibodies that target and initiate damage to peripheral nerves. DH1 was found to display an alphaNeuAc(2-8)alphaNeuAc(2-3)betaGal branch bound to the tetraheptosyl backbone core of its lipooligosaccharide (LOS). The novel sialylation pattern was found to be dependent on the activity of a bifunctional sialyltransferase, Lic3B, which catalyzes the addition of both the terminal and subterminal sialic acid residues. Patient serum IgGs bind to DH1 LOS, and the reactivity is significantly influenced by the presence of sialylated glycoforms. The display by DH1, of a surface glycan that mimics the terminal trisaccharide portion of disialosyl-containing gangliosides, provides strong evidence for its involvement in the development of Fisher syndrome.


Subject(s)
Gangliosides/metabolism , Haemophilus influenzae/physiology , Miller Fisher Syndrome/microbiology , Molecular Mimicry , Adult , Carbohydrate Sequence , Humans , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , Molecular Sequence Data
17.
Biochemistry ; 46(1): 36-44, 2007 Jan 09.
Article in English | MEDLINE | ID: mdl-17198373

ABSTRACT

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.


Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Antigens, Bacterial/chemistry , Campylobacter jejuni/immunology , Gangliosides/immunology , Lipopolysaccharides/immunology , Animals , Antibodies, Bacterial/chemistry , Antibodies, Monoclonal/chemistry , Antigens, Bacterial/immunology , Binding Sites, Antibody , Campylobacter Infections/immunology , Campylobacter jejuni/chemistry , Cross Reactions , Gangliosides/chemistry , Guillain-Barre Syndrome/immunology , Humans , Kinetics , Ligands , Lipopolysaccharides/chemistry , Magnetic Resonance Spectroscopy , Mice , Miller Fisher Syndrome/immunology , Surface Plasmon Resonance
18.
FEBS J ; 273(17): 3975-89, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16879613

ABSTRACT

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.


Subject(s)
Amides/chemistry , Bacterial Capsules/chemistry , Campylobacter jejuni/chemistry , Hyaluronic Acid/chemistry , Phosphoric Acids/chemistry , Sorbose/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Computer Simulation , Magnetic Resonance Spectroscopy , Models, Molecular , Spectrometry, Mass, Electrospray Ionization
19.
J Bacteriol ; 188(7): 2427-34, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16547029

ABSTRACT

In eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated that Campylobacter jejuni is the first member of the Bacteria to possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitate C. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation in Bacteria and provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.


Subject(s)
Bacterial Proteins/metabolism , Campylobacter jejuni/metabolism , Polysaccharides/biosynthesis , Polysaccharides/chemistry , Animals , Bacterial Proteins/genetics , Campylobacter Infections/microbiology , Campylobacter Infections/veterinary , Campylobacter jejuni/genetics , Chickens , Glycosylation , Lectins , Mutation , Nuclear Magnetic Resonance, Biomolecular , Poultry Diseases/microbiology
20.
J Biol Chem ; 281(17): 11480-6, 2006 Apr 28.
Article in English | MEDLINE | ID: mdl-16481326

ABSTRACT

We have identified a sialate O-acetyltransferase in the lipo-oligosaccharide biosynthesis locus of Campylobacter jejuni. Strains possessing this locus are known to produce sialylated outer core structures that mimic host gangliosides, and have been implicated in triggering the onset of Guillain-Barré syndrome. The acetyltransferase, which was cloned and expressed as a fusion construct in Escherichia coli, is soluble and homologous with members of the NodL-LacA-CysE family of O-acetyltransferases. This enzyme catalyzes the transfer of O-acetyl groups onto oligosaccharide-bound sialic acid, with a high specificity for terminal alpha2,8-linked residues. The modification is directed to C-9 and not C-7 as is believed to occur more commonly in other organisms. Despite their wide prevalence and importance in both eukaryotes and prokaryotes, this is the first report to describe the characterization of a purified sialate O-acetyltransferase.


Subject(s)
Acetyltransferases/metabolism , Campylobacter jejuni/enzymology , N-Acetylneuraminic Acid/metabolism , Oligosaccharides/metabolism , Acetyltransferases/genetics , Acetyltransferases/isolation & purification , Amino Acid Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Magnetic Resonance Spectroscopy , Molecular Sequence Data , N-Acetylneuraminic Acid/chemistry , Open Reading Frames , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid
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