title: Characterization and scope of S-layer protein O-glycosylation in Tannerella forsythia.
abstract: Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacterium's recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH(2)-(1→3)-[Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4-Me-α-Galp-(1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the three-amino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology, Vienna Institute of BioTechnology, Universität für Bodenkultur Wien, Muthgasse 11, A-1190 Vienna, Austria.
authors.forename: Gerald
authors.initials: G
authors.lastname: Posch
authors.affiliation: Department of Chemistry, Vienna Institute of BioTechnology, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.affiliation: Institute for Organic Chemistry, Universität Wien, Währingerstrasse 38, A-1090 Vienna, Austria.
authors.forename: Lothar
authors.initials: L
authors.lastname: Brecker
authors.affiliation: Department of Chemistry, Vienna Institute of BioTechnology, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology, Vienna Institute of BioTechnology, Universität für Bodenkultur Wien, Muthgasse 11, A-1190 Vienna, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Messner
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology, Vienna Institute of BioTechnology, Universität für Bodenkultur Wien, Muthgasse 11, A-1190 Vienna, Austria. Electronic address: christina.schaeffer@boku.ac.at.
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
mesh_terms: D001439:Bacteroides / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D016680:Genome, Bacterial; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry*; D009005:Monosaccharides / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013499:Surface Properties; D053719:Tandem Mass Spectrometry / Q000379:methods; D014774:Virulence
chemical_list: D006020:Glycopeptides; D008562:Membrane Glycoproteins; D009005:Monosaccharides; C055764:S-layer proteins