ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury

Abstract

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.

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

“…This followed our earlier work showing that ExoU was responsible for the acute cytotoxicity of cytotoxic strains of P. aeruginosa towards mammalian cells in vitro (Finck-Barbancon et al, 1997;Fleiszig et al, 1997). In this study we show that the mechanism for ExoU-mediated cytotoxicity towards corneal epithelial cells in vitro, and its promotion of ocular colonization and corneal disease pathology by 48 h in vivo, requires the phospholipase activity of this toxin.…”

The prophylactic effects of human IgG derived from sera containing high anti-PcrV titers against pneumonia-causingPseudomonas aeruginosa

“…PA103 came originally from a patient in Australia in the late 1960s, and is a cytotoxic strain with the exoS ¡ , exoT C , exoU C , and exoY C type III secretion system genotype, but has a negative ExoY secretion phenotype by virtue of a premature stop codon mutation. [50][51][52] PA103, stored as individual bacterial stocks at ¡80 C, was prepared for the experiments as described previously. 53,54 Briefly, bacterial cultures were grown at 33 C for 13 h in a shaking incubator, and then centrifuged at 8,500 £ g for 5 min.…”

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis

“…Both T2SS and the T3SS play independent roles in death due to Pseudomonas lung infection and are related to disease severity [18]. T3SS also enhances infection severity in several animal models: acute pneumonia [19,21,52], keratitis [52], bacteremia [22], peritonitis [53], burn infections [20] and gut-derived sepsis in neutropenia [54]. Moreover, T3SS in vitro detection was correlated with increased morbi-mortality and relapse in human P. aeruginosa ventilator-associated pneumonia and blood stream infections [55,56].…”