EXOU EXPRESSION BY PSEUDOMONAS-AERUGINOSA CORRELATES WITH ACUTE CYTOTOXICITY AND EPITHELIAL INJURY

The production of exoenzyme S is correlated with the ability of Pseudo monas 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 ag gregate 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 p ossess exoT but lack exoS, suggesting that ExoS is not the cytotoxin r esponsible 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 obs erved pathology. To identify the protein associated with acute cytotox icity, we compared extracellular protein profiles of PA103, its isogen ic non-cytotoxic derivative PA103 exsA::Omega and several cytotoxic an d non-cytotoxic P. aeruginosa clinical isolates, This analysis indicat ed that, in addition to expression of ExoT, expression of a 70-kDa pro tein correlated with the cytotoxic phenotype. Specific antibodies to t he 70-kDa protein bound to extracellular proteins from cytotoxic isola tes but failed to bind to similar antigen preparations from non-cytoto xic strains or PA103 exsA::Omega. To clone the gene encoding this pote ntial cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expr essed the 70-kDa extracellular protein but maintained expression of Ex oT. PA103 exoU::Tn5Tc was non-cytotoxic and failed to injure the epith elium in an acute lung infection model. Complementation of PA103exoU:: Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, Exo S and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with the ir co-ordinate regulation, In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino term inal motif that may be involved in the recognition of the type III sec retory apparatus of P. aeruginosa.