PILUS-FACILITATED ADHERENCE OF NEISSERIA-MENINGITIDIS TO HUMAN EPITHELIAL AND ENDOTHELIAL-CELLS - MODULATION OF ADHERENCE PHENOTYPE OCCURS CONCURRENTLY WITH CHANGES IN PRIMARY AMINO-ACID-SEQUENCE AND THE GLYCOSYLATION STATUS OF PILIN

Adherence of capsulate Neisseria meningitidis to endothelial and epith elial cells is facilitated in variants that express pill. Whereas pili ated variants of N. meningitidis strain C311 adhered to endothelial ce lls in large numbers (>150 bacteria/cell), derivatives containing spec ific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (<1 bacterium/c ell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Varian ts of strain C311 were obtained that expressed pilins of reduced appar ent M(r) and exhibited a marked increase in adherence to epithelial ce lls. Structural analysis of pilins from two hyper-adherent variants an d the parent strain were carried out by DNA sequencing of their pilE g enes. Deduced molecular weights of pilins were considerably lower comp ared with their apparent M(r) values on SDS-PAGE. Hyper-adherent pilin s shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at res idues 127-130 in mature pilin. Asn residues 113 and 127 of 'parental' pilin both form part of the typical eukaryotic N-glycosylation motif A sn-X-Ser/Thr and could potentially be glycosylated post-transiationall y. The presence of carbohydrate on pilin was demonstrated and when pil ins were deglycosylated, their migration on SDS-PAGE increased, suppor ting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pili ns produced by two fully piliated variants of a second strain (MC58) d iffered only in that the putative glycosylation motif Asn-60-Asn-61-Th r62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We pr opose, therefore, that functional variations in N. meningitidis pili m ay be modulated in large part by primary amino acid sequence changes t hat ablate or create N-linked glycosylation sites on the pilin subunit .