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.

A direct NMR method for the measurement of competitive kinetic isotope effects.

We present a technique that uses (13)C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (k(cat)/K(m)) for enzyme-catalyzed reactions. Using only milligram quantities of isotopically labeled substrates, precise competitive KIEs can be determined while following the ongoing reaction directly in a NMR spectrometer. Our results for the Vibrio cholerae sialidase-catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.

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.

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis.

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni alpha-(2-->3)-sialyltransferase (CST-06) and beta-(1-->4)-N-acetylgalactosaminyltransferase (CJL-30).

Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme.

Glycoconjugates containing polysialic acid have many biological activities and represent target molecules for therapeutic interventions. Enzymatic synthesis of these glycoconjugates should give access to these important molecules to evaluate their potential. The polysialyltransferases from both Neisseria meningitidis and Escherichia coli were cloned and expressed as recombinant proteins in E.

Variants of the beta 1,3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities.

The gene clusters encoding the lipooligosaccharide biosynthesis glycosyltransferases from Campylobacter jejuni have previously been divided in eight classes based on their genetic organization. Here, three variants of the beta1,3-galactosyltransferase CgtB from two classes were purified as fusions with the maltose-binding protein (MalE) from Escherichia coli and their acceptor preference was determined. The acceptor preference of each CgtB variant was directly related to the presence or absence of sialic acid in the acceptor, which correlated with the core oligosaccharide structure in vivo.

Structural characterization of Campylobacter jejuni lipooligosaccharide outer cores associated with Guillain-Barre and Miller Fisher syndromes.

Molecular mimicry between lipooligosaccharides (LOS) of Campylobacter jejuni and gangliosides in peripheral nerves plays a crucial role in the pathogenesis of C. jejuni-related Guillain-Barré syndrome (GBS). We have analyzed the LOS outer core structures of 26 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.

Evidence for acquisition of the lipooligosaccharide biosynthesis locus in Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, by horizontal exchange.

Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, has been shown to be genetically closely related to the completely sequenced strain C. jejuni NCTC 11168 by various molecular typing and serotyping methods. However, we observed that the lipooligosaccharide (LOS) biosynthesis genes strongly diverged between GB11 and NCTC 11168.

High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli.

The CMP-sialic acid synthetase (CMP-Neu5Ac, synthetase) is responsible for the synthesis of CMP-Neu5Ac, which is the donor used by sialyltransferases to attach sialic acid to acceptor hydroxyl groups in various polysaccharides, glycolipids, and glycoproteins. Since CMP-Neu5Ac is unstable and relatively expensive, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialylated oligosaccharides. We made a construct to over-express the Neisseria meningitidis CMP-Neu5Ac synthetase in Escherichia coli.

The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide.

We have compared the lipo-oligosaccharide (LOS) biosynthesis loci from 11 Campylobacter jejuni strains expressing a total of 8 different ganglioside mimics in their LOS outer cores. Based on the organization of the genes, the 11 corresponding loci could be classified into three classes, with one of them being clearly an intermediate evolutionary step between the other two. Comparative genomics and expression of specific glycosyltransferases combined with in vitro activity assays allowed us to identify at least five distinct mechanisms that allow C.