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J. Biol. Chem., Vol. 278, Issue 25, 22650-22656, June 20, 2003

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Proteolysis of Enteric Cell Villin by Entamoeba histolytica Cysteine Proteinases^*

Tineke Lauwaet  , Maria José Oliveira  ¶, Bert Callewaert , Georges De Bruyne  , Xavier Saelens || **, Serge Ankri  , Peter Vandenabeele ||, David Mirelman , Marc Mareel  and Ancy Leroy  ¶¶

From the Laboratory of Experimental Cancerology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium, the ^||Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, K. L. Ledeganckstraat 35, Ghent University, B-9000 Ghent, Belgium, and the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.

Received for publication, January 7, 2003 , and in revised form, April 2, 2003.

^* This work was supported by "de Belgische Federatie tegen Kanker." Work done at the Weizmann Institute was supported by the Center for Emerging Diseases (Jerusalem, Israel). Research in the Molecular Signaling and Cell Death Unit was supported by funding from the Flanders Interuniversity Institute for Biotechnology (VIB), the Interuniversitaire Attractiepolen V, the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Grant 3G.0006.01), the Bijzonder Onderzoeksfonds, the Geconcerteerde Onderzoeksacties (GOA), and European Union Research, Technological Development, and Demonstration Grant QLRT-1999-00739. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by GOA 2002 from Ghent University.

^¶ Recipient of a scholarship from the Portuguese Foundation for Science and Technology BD-15980.

^** Supported by the Biotech Fonds.

Present address: Dept. of Microbiology, Faculty of Medicine, Technion, Haifa 31096, Israel.

^¶¶ Postdoctoral fellow with the FWO-Vlaanderen, Belgium. To whom correspondence should be addressed. Tel.: 32-9-240-30-63; Fax: 32-9-240-49-91; E-mail: ancy.leroy{at}rug.ac.be.

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