Identification of functional regions of the Pseudomonas aeruginosa cytotoxin ExoU

Studies of the Pseudomonas aeruginosa cytotoxin ExoU have focused predominantly on its role in causing acute infections. Relatively little, however, is known about its mechanism of action. In particular, the contribution of different regions of ExoU to cytotoxicity is not well understood. By dissecting the roles of each region, we will better understand the mechanism by which ExoU causes cell death and thereby increases the severity of P. aeruginosa infections.; Most analyses have studied ExoU in the context of the mammalian tissue culture cell, but these experiments have been limited by the lack of a manipulable genetic system. The budding yeast Saccharomyces cerevisiae has many advantages for studying bacterial virulence factors, including genetic tractability, a fully sequenced genome, and regulation of exogenous genes. In this study, yeast were shown to be susceptible to ExoU-mediated killing and similar regions of ExoU were required for cytotoxicity to yeast and mammalian cells. These results suggest a similar mechanism of action and validate yeast as a model system for the study of ExoU. The yeast model and a transposon-based linker insertion mutagenesis technique followed by site-directed mutagenesis were used to further define the key regions and residues necessary for ExoU-induced cell death. Five regions were characterized and shown to be essential for both cytotoxicity and phospholipase A2 activity to both yeast and HeLa cells. These data indicate that phospholipase activity and cytotoxicity are directly correlated and that multiple regions of ExoU are required for both activities.; Although every region was required for phospholipase A2 activity, only four of these regions fall within a patatin-like phospholipase domain contained within the N-terminal half of ExoU. Examination of the fifth region revealed the C-terminal half to be both necessary and sufficient to target ExoU to the periphery of eukaryotic cells. These data suggest that peripheral localization is an essential step in the mechanism of ExoU-mediated cytotoxicity.