title: O-linked glycosylation ensures the normal conformation of the autotransporter adhesin involved in diffuse adherence.
abstract: The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. In this study, we identified several peptides of AIDA-I modified by the addition of heptoses by use of mass spectrometry and N-terminal sequencing of proteolytic fragments of AIDA-I. One threonine and 15 serine residues were identified as bearing heptoses, thus demonstrating for the first time that AIDA-I is O-glycosylated. We observed that unglycosylated AIDA-I is expressed in smaller amounts than its glycosylated counterpart and shows extensive signs of degradation upon heat extraction. We also observed that unglycosylated AIDA-I is more sensitive to proteases and induces important extracytoplasmic stress. Lastly, as was previously shown, we noted that glycosylation is required for AIDA-I to mediate adhesion to cultured epithelial cells, but purified mature AIDA-I fused to GST was found to bind in vitro to cells whether or not it was glycosylated. Taken together, our results suggest that glycosylation is required to ensure a normal conformation of AIDA-I and may be only indirectly necessary for its cell-binding function.
authors.affiliation: Université de Montréal, Faculté de Médecine Vétérinaire, 3200 Sicotte, St.-Hyacinthe, Québec J2S 7C6, Canada.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Charbonneau
authors.forename: Victoria
authors.initials: V
authors.lastname: Girard
authors.forename: Anastasia
authors.initials: A
authors.lastname: Nikolakakis
authors.forename: Manuel
authors.initials: M
authors.lastname: Campos
authors.forename: Frédéric
authors.initials: F
authors.lastname: Berthiaume
authors.forename: France
authors.initials: F
authors.lastname: Dumas
authors.forename: François
authors.initials: F
authors.lastname: Lépine
authors.forename: Michael
authors.initials: M
authors.lastname: Mourez
mesh_terms: D018830:Adhesins, Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D001422:Bacterial Adhesion / Q000502:physiology*; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006539:Heptoses / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
chemical_list: C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D006539:Heptoses; D010455:Peptides; D013912:Threonine; D012694:Serine; D010447:Peptide Hydrolases