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J. Bacteriol. doi:10.1128/JB.00864-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Proteolytic Processing is not Essential for Multiple Functions of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)

Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, 3200 Sicotte, St-Hyacinthe, J2S 7C6, Québec, Canada

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

	Abstract

The Escherichia coli Adhesin Involved in Diffused Adherence (AIDA-I), like many other autotransporter proteins, is released in the periplasm as a proprotein undergoing a 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 non-covalently 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 co-solubilized and co-purified 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 only be disturbed 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, auto-aggregation 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.

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