Aeromonas Exoenzyme T of Aeromonas salmonicida Is a Bifunctional Protein That Targets the Host Cytoskeleton

Michel R Popoff,Maryse Gibert,Joachim Frey,Sarah E Burr,Désirée Fehr,Jacques D'Alayer

doi:10.1074/jbc.m704797200

Michel R Popoff, Maryse Gibert + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m704797200

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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.

Highlights

To date, the following genes encoding four type III effector proteins have been identified in A. salmonicida: aexT, aopP, aopO, and aopH (8 –10)
We identify actin as the only specific target of the APD-ribosylating activity of Aeromonas exoenzyme T (AexT), and we show that AexT exhibits GTPase-activating proteins (GAP) activity against the monomeric GTPases Rho, Rac, and Cdc42
The results indicate that AexT exhibits specific ADP-ribosylating activity against actin, with the reaction against nonmuscular actin being significantly stronger than that against muscular actin (Fig. 1B, lanes 1 and 2)

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains—Cloning was routinely carried out in Escherichia coli strain XL1-Blue [14]. E. coli strain BL21(DE3) [15] was used for the expression of recombinant AexT proteins, and strain S17-1 [16] was used as the donor strain in bacterial conjugation. The wild type (WT) isolate A. salmonicida strain JF2267 [8] used in this study was routinely cultured on LuriaBertani agar plates at 18 °C. Site-directed Mutagenesis—The WT aexT gene was amplified from A. salmonicida strain JF2267 by PCR using primers aexTHIS-forward and aexTHIS-reverse (Table 1) or aexTGFPforward and aexTGFP-reverse (Table 1) before being cloned into the vector pGEM-T Easy (Promega). Site-directed mutagenesis via the overlap extension-PCR method [17] was

Oligonucleotide primers

Targeted mutation

Iota toxin

RESULTS

DISCUSSION