Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure.
Neisseria 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 'top-down' 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.
Aas
Finn Erik
FE
Centre for Molecular Biology and Neuroscience, Unversity of Oslo, 0316 Oslo, Norway.
Vik
Ashild
A
Vedde
John
J
Koomey
Michael
M
Egge-Jacobsen
Wolfgang
W
D000107:Acetylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D016123:Campylobacter jejuni / Q000378:metabolism; D036781:Fimbriae Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D005816:Genetic Complementation Test; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry*; D014312:Trisaccharides / Q000378:metabolism
D001426:Bacterial Proteins; D011134:Polysaccharides; D014312:Trisaccharides; C074063:pilE protein, Neisseria gonorrhoeae; D036781:Fimbriae Proteins; D016695:Glycosyltransferases