Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacterjejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide

N-acetyl neuraminic acid (NANA) is a common constituent of Campylobacter je juni lipo-oligosaccharide (LOS). Such structures often mimic human ganglios ides and are thought to be involved in the triggering of Guillain-Barre syn drome (GBS) and Miller-Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three puta tive NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA syntheta se activity of all three C. jejuni neuB gene products was confirmed by comp lementation experiments in an Escherichia coli neuB-deficient strain. Isoge nic mutants were created in all three neuB genes, and for one such mutant ( neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild- type LOS bound cholera toxin, indicating the presence of NANA in a LOS stru cture mimicking the ganglioside GM(1). This property was lost in the neuB1 mutant. Gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry analysis of LOS from wild-type and the neuB1 mutant strain de monstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis thro ugh condensation of N-acetyl-d-mannosamine and phosphoenolpyruvate. Souther n analysis demonstrated that the neuB1 gene was confined to strains of C. j ejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and n euB3 did not affect LOS, but neuB3 mutants were aflagellate and non-motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for s train G1 the flagellin protein from the neuB2 mutant showed an apparent red uction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distin ct surface structures: LOS and flagella.