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O-Linked Glycosylation Ensures the Normal Conformation of the Autotransporter Adhesin Involved in Diffuse Adherence

Marie-Ève Charbonneau,^1, Victoria Girard,^1, Anastasia Nikolakakis,² Manuel Campos,¹ Frédéric Berthiaume,¹ France Dumas,³ François Lépine,² and Michael Mourez^1*

Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, St.-Hyacinthe, 3200 Sicotte, St.-Hyacinthe, Québec J2S 7C6, Canada,¹ INRS-Institut Armand-Frappier, Université du Québec, 531 Boul. des Prairies, Laval, Québec H7V 1B7, Canada,² National Research Council, Biotechnology Research Institute, 6100 Royalmount, Montréal, Québec H4P 2R2, Canada³

Received 19 June 2007/ Accepted 6 October 2007

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.

Published ahead of print on 19 October 2007.

M.-È.C. and V.G. contributed equally to the work.