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Journal of Bacteriology, July 2001, p. 4330-4344, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4330-4344.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Multiple Domains Are Required for the Toxic Activity of Pseudomonas aeruginosa ExoU

Viviane Finck-Barbançon and Dara W. Frank*

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received 29 January 2001/Accepted 16 April 2001

Expression of ExoU by Pseudomonas aeruginosa is correlated with acute cytotoxicity in a number of epithelial and macrophage cell lines. In vivo, ExoU is responsible for epithelial injury. The absence of a known motif or significant homology with other proteins suggests that ExoU may possess a new mechanism of toxicity. To study the intracellular effects of ExoU, we developed a transient-transfection system in Chinese hamster ovary cells. Transfection with full-length but not truncated forms of ExoU inhibited reporter gene expression. Inhibition of reporter activity after cotransfection with ExoU-encoding constructs was correlated with cellular permeability and death. The toxicity of truncated versions of ExoU could be restored by coexpression of the remainder of the molecule from separate plasmids in trans. This strategy was used to map N- and C-terminal regions of ExoU that are necessary but not sufficient for toxicity. Disruption of a middle region of the protein reduces toxicity. This portion of the molecule is postulated to allow the N- and C-terminal regions to functionally complement one another. In contrast to ExoS and ExoT, native and recombinant ExoU molecules do not oligomerize or form aggregates. The complex domain structure of ExoU suggests that, like other P. aeruginosa-encoded type III effectors (ExoS and ExoT), ExoU toxicity may result from a molecule that possesses more than one activity.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Phone: (414) 456-8766. Fax: (414) 456-6535. E-mail: frankd{at}mcw.edu.

Journal of Bacteriology, July 2001, p. 4330-4344, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4330-4344.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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