Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion.

Pili, which are assembled from protein subunits called pilin, are indispensable for the adhesion of capsulated Neisseria meningitidis (MC) to eukaryotic cells. Both MC and Neisseria gonorrhoeae (GC) pilins are glycosylated, but the effect of this modification is unknown. In GC, a galactose alpha-1,3-N-acetyl glucosamine is O-linked to Ser-63, whereas in MC, an O-linked trisaccharide is present between residues 45 and 73 of pilin. As Ser-63 was found to be conserved in pilin variants from different strains, it was replaced by Ala in two MC variants to test the possible role of this residue in pilin glycosylation and modulation of pili function. The mutated alleles were stably expressed in MC, and the proteins they encoded migrated more quickly than the normal protein during SDS-PAGE. As controls, neighbouring Asn-61 and Ser-62 were replaced by an Ala with no effect on electrophoretic mobility. Silver staining of purified pilin obtained from MC after oxidation with periodic acid confirmed the loss of glycosylation in the Ser-63-->Ala pilin variants. Mass spectrometry of HPLC-purified trypsin-digested peptides of pilin and Ser-63-->Ala pilin confirmed that peptide 45-73 has the molecular size of a glycopeptide in the wild type. In strains producing non-glycosylated pilin variants, we observed that (i) no truncated S pilin monomer was produced; (ii) piliation was slightly increased; and (iii) presumably as a consequence, adhesiveness for epithelial cells was increased 1.6- to twofold in these derivatives. In addition, pilin monomers and/or individual pilus fibres, obtained after solubilization of a crude pili preparation in a high pH buffer, were reassociated into insoluble aggregates of pili more completely with non-glycosylated variants than with the normal pilin. Taken together, these data eliminate a major role for pilin glycosylation in piliation and subsequent pilus-mediated adhesion, but they demonstrate that glycosylation facilitates solubilization of pilin monomers and/or individual pilus fibres.