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.

Panton-Valentine leukocidin (PVL) is a Staphylococcus aureus (SA) exotoxin, which kills human granulocytes and monocytes in vitro. Among 43 SA strains from cutaneous infections, 12 were PVL producers, whereas among 49 blood culture strains, only 1 produced PVL. Most PVL-producing strains (11/22) came from 22 primitive cutaneous infections, especially furuncles and abscesses, while only 1 PVL-producing strain came from 21 secondary infections of dermatoses such as bullous or pruritic diseases. Intradermal injections of PVL in rabbits induced edema, erythema and necrosis; histopathological changes at the injection sites consisted mainly in leukocytoclasis and vascular necrosis. All changes were dose dependent, and previous immunization of rabbits partially neutralized PVL-induced effects. Production of PVL in vivo after injections of bacteria seems to be low, and the histopathological lesions induced in the rabbit skin do not appear to be specifically related to PVL activity. However, PVL is a good candidate as a new virulence factor in cutaneous SA infections.

Staphylococcus aureus ATCC 49775 produces three proteins recognized by affinity-purified antibodies against the S component of Panton-Valentine leucocidin (LukS-PV) and two proteins recognized by affinity-purified antibodies against the F component of this toxin (LukF-PV). Purification of these proteins and cloning of the corresponding genes provided evidence for the presence of two loci. The first one, encoding Panton-Valentine leucocidin, consisted of two cotranscribed open reading frames, lukS-PV and lukF-PV, coding the class S and the class F components, respectively. The second one coded for a gamma-hemolysin and consisted of two transcription units, the first one encoding an HlgA-like protein, a class S component, and the second one encoding two cotranscribed open reading frames identical to HlgC and HlgB, class S and class F components, respectively, from gamma-hemolysin from the reference strain Smith 5R. It appears that the Panton-Valentine leucocidin from S. aureus ATCC 49775 (V8 strain) should not be confused with leucocidin from ATCC 27733 (another isolate of V8 strain), which had 95% identity with HlgC and HlgB from gamma-hemolysin. The cosecretion of these five proteins led to six possible synergistic combinations between F and S components. Two of these combinations (LukS-PV-LukF-PV and HlgA-LukF-PV) had dermonecrotic activity on rabbit skin, and all six were leukocytolytic on glass-adsorbed leukocytes. Only three were hemolytic on rabbit erythrocytes, the two gamma-hemolysin combinations and the combination LukF-PV-HlgA.

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