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 indi spensable for the adhesion of capsulated Neisseria meningitidis (MC) t o 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, where as 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 fr om different strains, it was replaced by Ala in two MC variants to tes t the possible role of this residue in pilin glycosylation and modulat ion of pill function, The mutated alleles were stably expressed in MC, and the proteins they encoded migrated more quickly than the normal p rotein during SDS-PAGE. As controls, neighbouring Asn-61 and Ser-62 we re replaced by an Ala with no effect on electrophoretic mobility, Silv er staining of purified pilin obtained from MC after oxidation with pe riodic acid confirmed the loss of glycosylation in the Ser-63-->Ala pi lin variants, Mass spectrometry of HPLC-purified trypsin-digested pept ides 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 prod ucing non-glycosylated pilin variants, we observed that (i) no truncat ed S pilin monomer was produced; (ii) piliation was slightly increased ; and (iii) presumably as a consequence, adhesiveness for epithelial c ells was increased 1,6- to twofold in these derivatives, In addition, pilin monomers and/or individual pilus fibres, obtained after solubili zation of a crude pill preparation in a high pH buffer, were reassocia ted into insoluble aggregates of pill more completely with non-glycosy lated variants than with the normal pilin, Taken together, these data eliminate a major role for pilin glycosylation in piliation and subseq uent pilus-mediated adhesion, but they demonstrate that glycosylation facilitates solubilization of pilin monomers and/or individual pilus f ibres.