Nam Huan Khieu

Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans.

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

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. 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 β-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of d-glycero-α-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.

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)‐α‐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 β‐d‐fructofuranoses substituted at C‐3 with CH3OP(O)(NH2)(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 31P 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.

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)‐β‐d‐GlcA6NGro‐(1–3)‐β‐d‐GlcNAc‐(1‐]n repeating unit, where NGro is 2‐aminoglycerol. A labile α‐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.

CMP-pseudaminic acid is a natural potent inhibitor of PseB, the first enzyme of the pseudaminic acid pathway in Campylobacter jejuni and Helicobacter pylori.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and a significant cause of child morbidity in underdeveloped countries. There is also evidence linking C. jejuni infections to the development of Miller Fisher and Guillain-Barrneuropathies, the latter being the primary cause of neuroparalysis since the eradication of polio. Approximately two-thirds of the world’s population is infected with Helicobacter pylori, which is a major etiological agent of gastroduodenal disease and the only bacterium associated with cancer. As a result of the prevalence of infections caused by these pathogens and the increase in antibiotic resistant strains, novel therapeutics are urgently needed. Glycan biosynthetic pathways in bacteria are attractive therapeutic targets as many of these glycans are associated with cell-surface virulence factors and are unique to prokaryotes. C. jejuni and H. pylori decorate their flagella extensively with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-a-l-manno-nonulosonic acid or pseudaminic acid (Pse). Recently, C. jejuni was shown to decorate its flagellin with a number of structurally related nonulosonate derivatives as well. O-linked flagellin glycosylation with Pse is necessary for proper assembly of flagellar filaments, bacterial motility, colonization, and hence virulence. Agents that interfere with Pse production may therefore offer therapeutic potential. Recently, we identified six Pse biosynthesis enzymes (PseB, C, H, G, I, F, Figure 1) which constitute the complete CMP-Pse biosynthetic pathway starting from UDP-a-d-GlcNAc (1). 8] The initial enzyme, PseB, is considered unique as it is a 4,6-dehydratase/

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. 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.

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. We describe a unique and powerful biophysical technique applied to define the molecular interaction between autoimmune disease-causing antibodies and their ganglioside targets.

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 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.