Neisseria gonorrhoeae O‐linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Abstract

SummaryNeisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a 'topdown' mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di-and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O-and N-linked pathways can be combined in novel glycoengineering strategies.

Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae

“…The bacterial strains used in this study are described in Table S2 and were grown on conventional GC medium as described previously (15). Carboxyterminal 6xHis-tagged alleles as well as missense alleles of Ng1328 and Ng1371 were generated using PCR-based splicing by overlap extension reactions (29).…”

“…Purified PCR products were used to genetically transform Ngo strains (30), and correct transformants were screened for by PCR using diagnostic primer sets. Protein glycosylation null mutations were intro- duced into various strain backgrounds using transformation as previously described (15).…”

“…Trafficking to this compartment is likely a prerequisite for glycosylation because the PglO OTase is predicted to act in the periplasm (15,17). Potential functional connections can also be discerned among them.…”

“…Genetic analyses including interspecies complementation have revealed that this system (termed pgl for pilin glycosylation) is remarkably similar to the N-linked system of C. jejuni with regard to the use of a peptide-proximal 2,4 diacetamido-2,4,6 trideoxyhexose (DATDH) sugar and related biosynthetic pathways for generating lipid-linked glycan substrates (15). These systems then diverge at the step of oligosaccharide transfer with the Ser-targeting PglO OTase acting in Ngo and the sequon targeted PglB OTase in C. jejuni (supporting information Fig.…”

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].…”