SummaryThe production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::⍀ and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::⍀. To clone the gene encoding this potential cytotoxin we used Tn5 Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU :: Tn5 Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU ::Tn5 Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU ::Tn5 Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA 2 ) and cytosolic phospholipase A 2 (cPLA 2 ) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA 2 and cPLA 2 align or are similar to ExoU sequences. Sitespeci®c mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid pro®les and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modi®cation by eukaryotic factors.

The ability of Pseudomonas aeruginosa to secrete specific toxins using the type III-mediated pathway has been reported. To determine the association of this phenotype with human illness, immunoblot analysis was used to detect expression of type III secretory proteins in P. aeruginosa isolates from respiratory tract or blood cultures of 108 consecutive patients. Relative risk of mortality was 6-fold greater with expression of the type III secretory proteins ExoS, ExoT, ExoU, or PcrV. Phenotype was independently correlated with toxicity in cellular and murine models. Prevalence of this phenotype was significantly higher in acutely infected patients than in chronically infected patients with cystic fibrosis. These results suggest that the type III protein secretion system is integral to increased P. aeruginosa virulence. A positive phenotype is a predictor of poor clinical outcome. In the future, such analyses may help distinguish potentially lethal infection from colonization and help determine appropriate therapy for critically ill patients.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Damage to the lung epithelium is associated with the expression of toxins that are directly injected into eukaryotic cells through a type Ill-mediated secretion and translocation mechanism. Here we show that the P. aeruginosa homolog of the Yersinia V antigen, PcrV, is involved in the translocation of type III toxins. Vaccination against PcrV ensured the survival of challenged mice and decreased lung inflammation and injury. Antibodies to PcrV inhibited the translocation of type III toxins.

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