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Journal of Bacteriology, July 2001, p. 4330-4344, Vol. 183, No. 14
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.
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
*
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.
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