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.

Sialyltransferase inhibitors: consideration of molecular shape and charge/hydrophobic interactions.

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.

Gene function hypotheses for the Campylobacter jejuni glycome generated by a logic-based approach.

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.

Comparative analysis of photocaged RGDS peptides for cell patterning.

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).

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.

Lipooligosaccharide of Campylobacter jejuni: similarity with multiple types of mammalian glycans beyond gangliosides.

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.

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.

STD-NMR used to elucidate the fine binding specificity of pathogenic anti-ganglioside antibodies directly in patient serum.

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.

Glycosidase-induced fusion of isoprenoid gentiobiosyl lipid membranes at acidic pH.

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.

Complete chemoenzymatic synthesis of the Forssman antigen using novel glycosyltransferases identified in Campylobacter jejuni and Pasteurella multocida.

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.

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.

Campylobacter jejuni biofilms up-regulated in the absence of the stringent response utilize a calcofluor white-reactive polysaccharide.

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.

Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and 2H NMR.

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.

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni.

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.

A Haemophilus influenzae strain associated with Fisher syndrome expresses a novel disialylated ganglioside mimic.

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.

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.

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.

Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer.

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.

Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8-linked sialic acid.

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.

In vivo determination of Neisseria meningitidis serogroup A capsular polysaccharide by whole cell high-resolution magic angle spinning NMR spectroscopy.

High resolution-magic angle spinning (HRMAS) NMR spectroscopy was applied to serogroup A Neisseria meningitidis (NMA) to determine precise structures of capsular polysaccharide (CPS) expressed on the meningococcal surface. Both the O-acetylated (OAc) NMA parent and a mynC::aphA3 OAc- mutant demonstrated characteristic CPS-derived NMR signals indicating cell-surface expression of CPS, but only the parent expressed O-3 and O-4 acetylation signals. A capsule-defective strain showed no NMR signals for CPS.

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.

DRAMAtic transforms in magic angle spinning recoupling NMR: The Bessel function pathway.

In magic angle spinning (MAS) NMR recoupling experiments, the extraction of multiple couplings or a coupling distribution from the observed dephasing signals remains a challenging problem. At least for REDOR experiments, the REDOR transform solves this problem, enabling the simultaneous measurement of multiple dipolar couplings. Focusing on the quadrupolar dephasing observed in QUADRAMA experiments as a representative example, we demonstrate that the same analytical form used for the mathematical description of REDOR dephasing also describes the dephasing observed in a wide variety of MAS NMR recoupling experiments.

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.

Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses.

We recently demonstrated that Campylobacter jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system distinguishing Campylobacter into >60 groups. Although the wide variety of C. jejuni serotypes are suggestive of structural differences in CPS, the genetic mechanisms of such differences are unknown.