Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissueis considered to induce cross-reactive antibodies that lead to Guillain-Barré syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.
The structure and specificity of the recombinant a-2,3-sialyltransferase from Neisseria meningitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use a-terminal and pterminal Gal residues as acceptors. In addition (pl-+4)-linked and (pl+3)-linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane-associated protein in Escherichia coli at a level of 750 U/I (-250 mgfl). The protein could be extracted with buffers containing 0.2% Triton X-100 and purified to homogeneity using immobilized-metal-affinity chromatography. Electrospray-ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95 % of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP-NeuSAc synthetase, the a-2,3-sialyltransferase could sialylate fluorescent derivatives of N-acetyllactosamine with N-acetylneuraminic acid, N-propionylneuraminic acid and N-glycoloylneuraminic acid.Keywords: Neisseria meningitidis ; sialyltransferase Sialyltransferases are a group of glycosyltransferases that transfer sialic acid (N-acetylneuraminic acid, NeuSAc) from the activated sugar nucleotide CMP-NeuSAc to acceptor oligosaccharides found on glycoproteins, glycolipids or polysaccharides. It has become apparent that these sialylated oligosaccharides play important roles in cell-cell recognition, cell differentiation, and various receptor-ligand interactions in mammalian systems. The large number of sialylated oligosaccharide structures has led to the characterization of many sialyltransferases involved in the synthesis of these structures. Based on the linkage and acceptor specificity of the sialyltransferases studied, it has been determined that at least 13 sialyltransferase genes are present in mammalian systems [l].Sialylated glycoconjugates are also found in bacteria [2-31, where they are structures that mimic oligosaccharides found in mammalian glycolipids [4] and are probably used to evade the host immune response. The importance of sialylated lipo-oligosaccharides (LOS) in the pathogenesis of Neisseria gonorrhoeae has been established [5], while for Neisseria meningitidis the polysialic acid capsule and the sialylated LOS were found to be important for pathogenicity [6]. Despite their importance as proven or potential virulence factors, few bacterial sialyltransferases have been cloned [7-91 or purified [lo]. The a-2,8-sialyltransferases involved in the synthesis of the polysialic acid capsules have been cloned and expressed from both Escherichia coli [7] and N. meningitidis [S], and we have recently cloned the LOS a-2,3-sialyltransferases from N. meningitidis and N. gonorrhoeae [9].Because of the biological activity of their products, mammalian sialyltransferases are expected to act in specific tissues, cell compartments and/or developmental stages to create precise sialyloglycans. Bact...
The recent cloning of the lipooligosaccharide (LOS) A-2,3-sialyltransferase from Neisseria meningitidis immunotype L3 permitted us to examine other immunotypes for this structural gene. We identified the gene and measured the enzyme activity in the L1 immunotype strain which had previously been reported to lack sialic acid in its LOS because it contains a terminal A-linked galactose which was thought not to be an acceptor for the sialyltransferase. This finding prompted us to re-examine the structure of the LOS from the L1 immunotype, which revealed the presence of sialic acid on the terminal A-linked galactose. Oligosaccharides derived from the LOS were shown to be sialylated by composition and methylation analysis, mass spectrometry and nuclear magnetic resonance. The detailed structural analysis showed the sialic acid to occur only at O6 of the teminal A-D-galactopyranose residue of the A-D-Gal-1,4-β-D-Gal-1,4-β-D-glc trisaccharide (P k epitope) chain of the LOS, in the A-D configuration. These data are the first report of a A-2,6-linked sialic acid in a bacterial LOS or lipopolysaccharide, and also the first report of a sialylated P k epitope.Keywords : sialyltransferase ; Neisseria meningitidis; glycosyltransferase; lipooligosaccharide.The A-2,3-sialyltransferase involved in the biosynthesis of The mucosal pathogens in the genera Neisseria, Haemophilus, Moraxella, Campylobacter and Bordetella all possess a ma-sialyllacto-N-neotetraose has been reported to occur in the major cell surface lipooligosaccharide (LOS) which has features jority of the N. meningitidis immunotypes as well as N. gonorthat distinguish it from the enterobacterial lipopolysaccharide rhoeae isolates [8]. The distibution of enzyme activity does not (LPS) [1]. Perhaps the most important distinction is that struc-always correlate with the presence or absence of lacto-N-neotures displayed in LOS from these bacteria have some structural tetraose in the LOS. We previously reported that the N. meninsimilarity to human glycolipids. The LOS molecules elaborated gitidis A-2,3-sialyltransferase has a relaxed acceptor specificity by strains of N. meningitidis and N. gonorrhoeae that have been in that it would use synthetic acceptors which presented terminal shown to be important in the disease mechanism include those N-acetyllactosamine, lactose or galactose [9]. We have extended presenting the following oligosaccharide structures: Lacto-N-our examination of this enzyme and have reported recently that neotetraose, Sialyllacto-N-neotetraose, and the P k blood group the A-2,3-sialyltransferase from N. meningitidis immunotypes L3 antigen [1] (Fig. 1). These oligosaccharides provide these patho-and L1 can also use the terminal A-D-galactose, to make an Agens with a means of evading the host immune response through 2,3-sialyl-P k -trisaccharide in vitro with a synthetic acceptor molmolecular mimicry and are therefore potent virulence factors [2]. ecule [10]. Since we had demonstrated that an A-D-galactose resThe genetics of LPS biosynthesis have been ext...
Human influenza viruses are proposed to recognize sialic acids (pink diamonds) on glycans extended with poly‐LacNAc chains (LacNAc=(yellow circle+blue square)). N‐ and O‐linked glycans were extended with different poly‐LacNAc chains with α2‐3‐ and α2‐6‐linked sialic acids recognized by human and avian influenza viruses, respectively. The specificity of recombinant hemagglutinins (receptors in green) was investigated by using glycan microarray technology.
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