title: Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases.
abstract: The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.
authors.affiliation: Centre for Molecular Biology and Neuroscience, Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Finn Erik
authors.initials: FE
authors.lastname: Aas
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Egge-Jacobsen
authors.forename: Hanne C
authors.initials: HC
authors.lastname: Winther-Larsen
authors.forename: Cecilia
authors.initials: C
authors.lastname: Løvold
authors.forename: Paul G
authors.initials: PG
authors.lastname: Hitchen
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Michael
authors.initials: M
authors.lastname: Koomey
mesh_terms: D000595:Amino Acid Sequence; D004982:Ethanolaminephosphotransferase / Q000502:physiology*; D004983:Ethanolamines / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000378:metabolism*; D010767:Phosphorylcholine / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
chemical_list: D004983:Ethanolamines; D008070:Lipopolysaccharides; C074063:pilE protein, Neisseria gonorrhoeae; D010767:Phosphorylcholine; D036781:Fimbriae Proteins; C005448:phosphorylethanolamine; D004982:Ethanolaminephosphotransferase