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J. Bacteriol. doi:10.1128/JB.00969-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

O-linked glycosylation ensures the normal conformation of the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)

Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, St-Hyacinthe, 3200 Sicotte, St-Hyacinthe, Québec, J2S 7C6, Canada. INRS-Institut Armand-Frappier, Université du Québec, 531 Boul. des Prairies, Laval, Québec, H7V 1B7, Canada. National Research Council, Biotechnology Research Institute, Publication #NRC47555, 6100 Royalmount, Montréal, Québec, H4P 2R2, Canada

^* To whom correspondence should be addressed. Email: m.mourez{at}umontreal.ca.

	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 using 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 the unglycosylated AIDA-I is more sensitive to proteases and induces an 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 is 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.