Genetic characterization of pilin glycosylation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host, pill are post-translationally modified by addition of an O-link ed trisaccharide, Gal(beta 1-4)Gal(alpha 1-3)2,4-diacetimido-2,4,6-trideoxy hexose. In a previous study the authors identified and characterized a gene , pglA, encoding a galactosyltransferase involved in pilin glycosylation. I n this study a set of random genomic sequences from N. meningitidis strain MC58 was used to search for further genes involved in pilin glycosylation. Initially, an open reading frame was identified, and designated pglD (pilin glycosylation gene D), which was homologous to genes involved in polysacch aride biosynthesis, The region adjacent to this gene was cloned and nucleot ide sequence analysis revealed two further genes, pglB and pglC, which were also homologous with genes involved in polysaccharide biosynthesis, Insert ional mutations were constructed in pglB, pglC and pglD in N, meningitidis C311#3, a strain with well-defined LPS and pilin-linked glycan structures, to determine whether these genes had a role in the biosynthesis of either o f these molecules. Analysis of these mutants revealed that there was no alt eration in the phenotype of LPS in any of the mutant strains as judged by S DS-PAGE gel migration. In contrast, increased gel migration of the pilin su bunit molecules of pglB, pglC and pglD mutants by Western analysis was obse rved, Pilin from each of the pglB, pglC and pglD mutants did not react with a terminal-galactose-specific stain, confirming that the gel migration dif ferences were due to the alteration or absence of the pilin-linked trisacch aride structure in these mutants. In addition, antisera specific for the C3 11#3 trisaccharide failed to react with pilin from the pglB, pglC, pglD and galE mutants. Analysis of nucleotide sequence homologies has suggested spe cific roles for pglB, pglC and pglD in the biosynthesis of the 2,4-diacetim ido-2,4,6-trideoxyhexose structure.