UC San Diego

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes severe infections in individuals with compromised immune systems. In cystic fibrosis patients, infection with P. aeruginosa is the leading cause of death. A number of highly virulent strains of P. aeruginosa have been found to express the cytotoxic protein ExoU. Correspondingly, ExoU has been shown to increase virulence in animal models of pneumonia. ExoU is transported by the P. aeruginosa type III secretion system directly into the cytosol of host cells, where it exerts cytotoxicity. At the start of the studies described in this dissertation, the mechanism of action of ExoU was unknown. Presented here are studies uncovering the mechanism of action of ExoU from P. aeruginosa. Evidence points to ExoU functioning as a host-cell activated phospholipase A2 enzyme. Mutagenesis in the putative catalytic site or the presence of phospholipase A2 inhibitors is found to block ExoU-induced cytotoxicity. Furthermore, cytotoxicity of an ExoU-expressing strain of P. aeruginosa is blocked by phospholipase A2 inhibitors, suggesting a novel mode of treatment for P. aeruginosa infections. ExoU has no in vitro phospholipase A2 activity without the presence of a cellular extract, suggesting that ExoU requires one or more host cell factors for activation. Genetic and biochemical experiments were carried out to determine the host cell factors involved in ExoU activation. Genetic experiments with yeast, which are found to be susceptible to ExoU-induced cytotoxicity, suggest that the gene required for ExoU activation may be essential for viability or have a redundant function. Biochemical purification of the ExoU activator from a yeast extract suggests that it is a heat-stable, anionic and glucose-containing saccharide. However, this molecule is unable to induce rapid activation of ExoU, as is seen in vivo, suggesting the involvement of another factor, termed the ExoU accelerator. The ExoU accelerator is found to be heat-labile and part of a very large complex containing RNA. Surprisingly, these studies show that the saccharide activator does not co-purify with this factor. This suggests that there may be two completely different ExoU activators, or that the saccharide activator is an unusual glycosylation modification on the ExoU accelerator