Genetic characterization of pilin glycosylation in Neisseria meningitidis The GenBank accession number for the sequence determined in this work is AF014804.

Abstract

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. Pili are post-translationally modified by addition of an O-linked trisaccharide, Gal(β1-4)Gal(α1-3)2,4-diacetimido-2,4,6-trideoxyhexose. In a previous study the authors identified and characterized a gene, pglA, encoding a galactosyltransferase involved in pilin glycosylation. In 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 polysaccharide biosynthesis. The region adjacent to this gene was cloned and nucleotide sequence analysis revealed two further genes, pglB and pglC, which were also homologous with genes involved in polysaccharide biosynthesis. Insertional mutations were constructed in pglB, pglC and pglD in N. meningitidis C311F3, a strain with well-defined LPS and pilin-linked glycan structures, to determine whether these genes had a role in the biosynthesis of either of these molecules. Analysis of these mutants revealed that there was no alteration in the phenotype of LPS in any of the mutant strains as judged by SDS-PAGE gel migration. In contrast, increased gel migration of the pilin subunit molecules of pglB, pglC and pglD mutants by Western analysis was observed. Pilin from each of the pglB, pglC and pglD mutants did not react with a terminalgalactose-specific stain, confirming that the gel migration differences were due to the alteration or absence of the pilin-linked trisaccharide structure in these mutants. In addition, antisera specific for the C311F3 trisaccharide failed to react with pilin from the pglB, pglC, pglD and galE mutants. Analysis of nucleotide sequence homologies has suggested specific roles for pglB, pglC and pglD in the biosynthesis of the 2,4-diacetimido-2,4,6-trideoxyhexose structure.

Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

“…Bacillosamine (Bac), a diaminotrideoxyhexose, has been found in many prokaryotes (36), most recently in Pseudomonas reactans (35). An N-linkage between Bac and Asn residues has not been reported before, although it is possible that an O-linkage of Bac to Ser is present in the pilin of N. meningitidis (5,6). The mass data reported for the O-linked sugar are consistent with Bac (38), but its stereochemistry has not been reported.…”

“…The pgl locus of C. jejuni is very similar to one in N. meningitidis that is responsible for the O-glycosylation of pilin (5,6), as shown in Fig. 8.…”

“…Removal of the glycan moieties by pgl mutation resulted in decreased adherence and invasion in vitro and loss of mouse colonization in vivo (4), suggesting that protein glycosylation influences the virulence properties of this organism. A similar genetic locus has recently been described for pilin O-glycosylation in Neisseria meningitidis (5,6).…”

Diversity, Abundance and Distribution of O-linked Glycosylation Pathway Enzymes in Prokaryotes-A Comparative Genomics Study

“…In the genus Neisseria, the O-glycosylation pathway ( Figure S1) has been delineated in the species gonorrhoeae [1,5], lactamica [15] and meningitidis [2,3]. The enzymes involved in these pathways have been characterized to various extents [1][2][3][4][15][16][17][18][19]. For example, in Neisseria meningitidis, PglE has been shown to be a β1,4-GalT and pglE has been shown to be responsible for phase variation between tri-and disaccharide structures [5].…”

“…These are the O-glycosylation pathways of Neisseria [1][2][3][4][5], Helicobacter pylori [6], Pseudomonas aeruginosa [7], Bacteroides fragilis [8] and Acinetobacter baumannii [9], and the N-glycosylation pathways of Campylobacter jejuni [10][11][12], Haloferax volcanii [13] and Methanococcus voltae [14]. In the genus Neisseria, the O-glycosylation pathway ( Figure S1) has been delineated in the species gonorrhoeae [1,5], lactamica [15] and meningitidis [2,3]. The enzymes involved in these pathways have been characterized to various extents [1][2][3][4][15][16][17][18][19].…”