Aeromonas Exoenzyme T of Aeromonas salmonicida Is a Bifunctional Protein That Targets the Host Cytoskeleton*
- Désirée Fehr‡,
- Sarah E. Burr‡,
- Maryse Gibert§,
- Jacques d'Alayer¶,
- Joachim Frey‡1 and
- Michel R. Popoff§
- ‡Institute of Veterinary Bacteriology, Universität Bern, Länggassstrasse 122, Postfach, CH-3001 Bern, Switzerland and §Unité des Bacteries Anaerobies et Toxines and ¶Plateforme d'Analyse et de Microsequençage des Protéines, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris Cedex 15, France
- 1 To whom correspondence should be addressed: Institute of Veterinary Bacteriology, Universität Bern, Länggasstrasse 122, Postfach, 3001 Bern, Switzerland. Tel.: 41-31-6312-414; Fax: 41-31-6312-634; E-mail: joachim.frey{at}vbi.unibe.ch.
Abstract
Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.
Footnotes
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↵2 The abbreviations used are: GAP, GTPase-activating protein; WT, wild type; EPC cells, epithelioma papulosum cyprinid cells; EGFP, enhanced green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; HPLC, high pressure liquid chromatography.
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↵* This work was supported by the Swiss National Science Foundation Grant SNF 3100A0-10159 and by the Institute Pasteur. 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.
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- Received June 11, 2007.
