Polymorphisms in pilin glycosylation locus of Neisseria meningitidis expressing class II pili

We have located a locus, pgl, in Neisseria meningitidis strain NMB required for the glycosylation of class II pili. Between five and eight open readin g frames (ORFs) (pglF, pglB, pglC, pglB2, orf2, orf3, orf8, and avtA) were present in the pgl clusters of different meningococcal isolates. The Class I pilus-expressing strains Neisseria gonorrhoeae MS11 and N. meningitidis M C58 each contain a pgl cluster in which orf2 and orf3 have been deleted. St rain NMB and other meningococcal isolates which express class II type IV pi ll contained pgl clusters in which pglB had been replaced by pglB2 and an a dditional novel ORF, orf8, had been inserted between pglB2 and pglC. Insert ional inactivation of the eight ORFs of the pgl cluster of strain NMB showe d that pgl2F, pglB2, pglC, and pglD, but not orf2, orf3, orf8, and avtA, we re necessary for pilin glycosylation. Pilin glycosylation was not essential for resistance to normal human serum, as pglF and pglD mutants retained wi ld-type levels of serum resistance. Although pglB2 and pglC mutants were si gnificantly sensitive to normal human serum under the experimental conditio ns used, subsequent examination of the encapsulation phenotypes revealed th at pglB2 and pglC mutants expressed almost 50% less capsule than wild-type NMB. A mutation in orf3, which did not affect pilin glycosylation, also res ulted in a 10% reduction in capsule expression and a moderately serum sensi tive phenotype. On the basis of these results we suggest that pilin glycosy lation may proceed via a lipid-linked oligosaccharide intermediate and that blockages in this pathway may interfere with capsular transport or assembl y.