Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Abstract

SummaryPili 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 either an O-linked trisaccharide, Gal ( b b b b 1-4) Gal ( a a a a 1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an Olinked disaccharide Gal ( a a a a 1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pglB/B2 , pglF, pglG and pglH . A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pglB2 polymorphisms were not found in strain C311#3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311#3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311#3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311#3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311#3 and other strains. We also present evidence that pglG, pglH and pglB2 are potentially phase variable.

Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue

“…PglB is a bifunctional enzyme, the carboxyl-terminal half (PglBCter) has the acetylation activity described above, and the amino-terminal half transfers the activated DATDH to a lipid carrier. Galactose residues are added to DATDH by pglA and pglE (19). The glycolipid would be transported to the periplasmic side of the inner membrane by PglF, and, finally, PglL would transfer the oligosaccharide to PilE (20).…”

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

“…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].…”

“…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].…”

“…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]. In Neisseria gonorrhoeae, enzyme activities, substrate specificities and steady state kinetics parameters have been determined [3]. Functional characterization of PglL from Neisseria meningitidis and PilO from Pseudomonas aeruginosa has shown that both these enzymes have relaxed glycan specificity and they require the glycan to be translocated to the periplasm [7].…”

“…In N. gonorrhoeae and N. meningitidis, the protein O-glycosylation enzymes are clustered and form the pilin glycosylation locus [20]. Pgl polymorphism, phase variability and competition among the enzymes for a common substrate may lead to glycoforms [3,17,20] i.e., variants of a glycoprotein which differ from each other only in the nature of attached glycan [21]. For example, strains which possess NsPglB1 have 2,4-diacetamido-2,4,6-trideoxy-glucose at the reducing end of the glycans; in contrast, strains which possess its variant allele NsPglB2 have 4-glyceramido-2-acetamido-2,4,6-trideoxy-hexosamine [22].…”