IDENTIFICATION OF A NOVEL GENE INVOLVED IN PILIN GLYCOSYLATION IN NEISSERIA-MENINGITIDIS

The pili of Neisseria meningitidis are a key virulence factor, being m ajor adhesins of this capsulate organism that contribute to specificit y for the human host. Recently it has been reported that meningococcal pill are post-translationally modified by the addition of an O-linked trisaccharide, Gal (beta 1-4) Gal (alpha 1-3) 2,4-diacetimido-2,4,6-t rideoxyhexose. Using a set of random genomic sequences from N, meningi tidis strain MC58, we have identified a novel gene homologous to a fam ily of glycosyltransferases. A plasmid clone containing the gene was i solated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined, The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mut ations were constructed in pglA in a range of meningococcal strains wi th well-defined lipopolysaccharide (LPS) or pilin-linked glycan struct ures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclona l antibodies, In contrast, decreased gel migration of the pilin subuni t molecules of pglA mutants was observed, which was similar to the mig ration of pilins of galE mutants of same strains, supporting the notio n that pglA is a glycosyltransferase involved in the biosynthesis of t he pilin-linked trisaccharide structure, The pglA mutation, like the g alE mutation reported previously, had no effect on pilus-mediated adhe sion to human epithelial or endothelial cells, Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (bet a 1-4) Gal structure, suggesting that PglA is a glycosyltransferase in volved in the addition of galactose of the trisaccharide substituent o f pilin.