Multiple domains are required for the toxic activity of Pseudomonas aeruginosa ExoU

Expression of ExoU by Pseudomonas aeruginosa is correlated with acute cytot oxicity in a number of epithelial and macrophage cell lines. In vivo, ExoU is responsible for epithelial injury. The absence of a known motif or signi ficant homology with other proteins suggests that ExoU may possess a new me chanism of toxicity. To study the intracellular effects of ExoU, we develop ed a transient-transfection system in Chinese hamster ovary cells. Transfec tion with full-length but not truncated forms of ExoU inhibited reporter ge ne expression. Inhibition of reporter activity after cotransfection with Ex oU-encoding constructs was correlated with cellular permeability and death. The toxicity of truncated versions of ExoU could be restored by coexpressi on of the remainder of the molecule from separate plasmids in trans. This s trategy was used to map N- and C-terminal regions of ExoU that are necessar y but not sufficient for toxicity. Disruption of a middle region of the pro tein reduces toxicity. This portion of the molecule is postulated to allow the N- and C-terminal regions to functionally complement one another. In co ntrast to ExoS and ExoT, native and recombinant ExoU molecules do not oligo merize or form aggregates. The complex domain structure of ExoU suggests th at, like other P. aeruginosa-encoded type III effecters (ExoS and ExoT), Ex oU toxicity may result from a molecule that possesses more than one activit y.