title: ExoU-induced procoagulant activity in Pseudomonas aeruginosa-infected airway cells.
abstract: The present study addressed the question whether ExoU, a Pseudomonas aeruginosa toxin with phospholipase A2 (PLA2) activity, may induce airway epithelial cells to overexpress tissue factor (TF) and exhibit a procoagulant phenotype. Cells from the human bronchial epithelial BEAS-2B line were infected with an ExoU-producing P. aeruginosa strain, pre-treated or not with the cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP), or with two ExoU-deficient mutants. Control noninfected and infected cells were assessed for the expression of: 1) TF mRNA by RT-PCR; 2) cell-associated TF by enzyme immunoassay and flow cytometry; 3) procoagulant activity by a colorimetric assay; and 4) microparticle-associated TF by flow cytometry. An enzyme immunoassay was also used to assess cell-associated TF in lung extracts from mice infected intratracheally with ExoU-producing and -deficient bacteria. Cells infected with the wild-type bacteria had higher levels of TF mRNA, cell-associated TF expression, procoagulant activity and released microparticle-associated TF than cells infected with the mutants. Bacterial treatment with MAFP significantly reduced the expression of TF by infected cells. Lung samples from mice infected with the wild-type bacteria exhibited higher levels of cell-associated TF and procoagulant activity. The present results demonstrate that ExoU may contribute to the pathogenesis of lung injury by inducing a tissue factor-dependent procoagulant activity in airway epithelial cells.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil. crisplot@yahoo.com.br
authors.forename: M C
authors.initials: MC
authors.lastname: Plotkowski
authors.forename: L F P
authors.initials: LF
authors.lastname: Feliciano
authors.forename: G B S
authors.initials: GB
authors.lastname: Machado
authors.forename: L G
authors.initials: LG
authors.lastname: Cunha
authors.forename: C
authors.initials: C
authors.lastname: Freitas
authors.forename: A M
authors.initials: AM
authors.lastname: Saliba
authors.forename: M C
authors.initials: MC
authors.lastname: de Assis
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001980:Bronchi / Q000166:cytology / Q000382:microbiology*; D003029:Coagulants / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D063065:Organophosphonates / Q000378:metabolism; D054467:Phospholipases A2 / Q000378:metabolism; D011552:Pseudomonas Infections / Q000175:diagnosis / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D001095:Arachidonic Acids; D001426:Bacterial Proteins; D003029:Coagulants; D063065:Organophosphonates; C104810:methyl arachidonylfluorophosphonate; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D013925:Thromboplastin; D054467:Phospholipases A2