Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton.
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.
Fehr
Désirée
D
Institute of Veterinary Bacteriology, Universität Bern, Länggassstrasse 122, Postfach, CH-3001 Bern, Switzerland and.
Burr
Sarah E
SE
Institute of Veterinary Bacteriology, Universität Bern, Länggassstrasse 122, Postfach, CH-3001 Bern, Switzerland and.
Gibert
Maryse
M
Unité des Bacteries Anaerobies et Toxines, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris Cedex 15, France.
d'Alayer
Jacques
J
Plateforme d'Analyse et de Microsequençage des Protéines, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris Cedex 15, France.
Frey
Joachim
J
Institute of Veterinary Bacteriology, Universität Bern, Länggassstrasse 122, Postfach, CH-3001 Bern, Switzerland and. Electronic address: joachim.frey@vbi.unibe.ch.
Popoff
Michel R
MR
Unité des Bacteries Anaerobies et Toxines, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris Cedex 15, France.
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D048409:Aeromonas salmonicida / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003530:Cyprinidae; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D013379:Substrate Specificity; D014709:Vero Cells; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029941:Fish Proteins; D036002:ADP Ribose Transferases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins