title: Proteolytic processing is not essential for multiple functions of the Escherichia coli autotransporter adhesin involved in diffuse adherence (AIDA-I).
abstract: The Escherichia coli adhesin involved in diffuse adherence (AIDA-I), like many other autotransporter proteins, is released in the periplasm as a proprotein undergoing proteolytic processing after its translocation across the outer membrane. The proprotein is cleaved into a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with the outer membrane and can be released by heat treatment. The mechanism of cleavage of the proprotein and its role in the functionality of AIDA-I are not understood. Here, we show that cleavage is independent of the amount of AIDA-I in the outer membrane, suggesting an intramolecular autoproteolytic mechanism or a cleavage mediated by an unknown protease. We show that the two fragments, mature AIDA-I and AIDAc, can be cosolubilized and copurified in a folded and active conformation. We observed that the release by heat treatment results from the unfolding of AIDA-I and that the interaction of AIDA-I with AIDAc seems to be disturbed only by denaturation. We constructed an uncleavable point mutant of AIDA-I, where a serine of the cleavage site was changed into a leucine, and showed that adhesion, autoaggregation, and biofilm formation mediated by the mutant are indistinguishable from the wild-type levels. Lastly, we show that both proteins can mediate the invasion of cultured epithelial cells. Taken together, our experiments suggest that the proteolytic processing of AIDA-I plays a minor role in the functionality of this protein.
authors.affiliation: Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, 3200 Sicotte, St-Hyacinthe, J2S 7C6, Québec, Canada.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Charbonneau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Berthiaume
authors.forename: Michael
authors.initials: M
authors.lastname: Mourez
mesh_terms: D018830:Adhesins, Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion; D018441:Biofilms / Q000254:growth & development; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational
chemical_list: C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli