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Journal of Bacteriology, November 2003, p. 6583-6591, Vol. 185, No. 22
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.22.6583-6591.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The ADP-Ribosylating Toxin, AexT, from Aeromonas salmonicida subsp. salmonicida Is Translocated via a Type III Secretion Pathway

Sarah E. Burr, Katja Stuber,{dagger} and Joachim Frey*

Institute of Veterinary Bacteriology, University of Berne, CH-3012 Berne, Switzerland

Received 14 February 2003/ Accepted 26 August 2003

AexT is an extracellular ADP ribosyltransferase produced by the fish pathogen Aeromonas salmonicida subsp. salmonicida. The protein is secreted by the bacterium via a recently identified type III secretion system. In this study, we have identified a further 12 open reading frames that possess high homology to genes encoding both structural and regulatory components of the Yersinia type III secretion apparatus. Using marker replacement mutagenesis of aopB, the A. salmonicida subsp. salmonicida homologue of yopB in Yersinia, we demonstrate that the bacterium translocates the AexT toxin directly into the cytosol of cultured fish cells via this type III secretion pathway. An acrV mutant of A. salmonicida subsp. salmonicida displays a calcium-blind phenotype, expressing and secreting significant amounts of AexT even in the presence of CaCl2 concentrations as high as 10 mM. This acrV mutant is also unable to translocate AexT into the cytosol of fish cells, indicating AcrV is involved in the translocation process. Inactivation of either the aopB or acrV gene in A. salmonicida subsp. salmonicida (resulting in an inability to translocate AexT) is accompanied by a loss of cytotoxicity that can be restored by trans complementation. Finally, we present data indicating that preincubation of the wild-type bacteria with antibodies directed against recombinant AcrV-His protein provides fish cells protection against the toxic effects of the bacterium.

* Corresponding author. Mailing address: Institute of Veterinary Bacteriology, University of Berne, Laenggassstrasse 122, Postfach, CH-3001 Berne, Switzerland. Phone: 41 31 6312 414. Fax: 41 31 6312 634. E-mail: joachim.frey{at}vbi.unibe.ch.

{dagger} Present address: Microbiological Laboratory, Swiss Federal Veterinary Office, Berne, Switzerland.

Journal of Bacteriology, November 2003, p. 6583-6591, Vol. 185, No. 22
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.22.6583-6591.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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