title: Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis.
abstract: BACKGROUND
  ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF) in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1).
  
  
  METHODS
  Mice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF) were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection.
  
  
  RESULTS
  In PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively.
  
  
  CONCLUSIONS
  ExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in vitro detection of exoU gene in bacterial clinical isolates warrants investigation as a predictor of outcome of patients with P. aeruginosa pneumonia/sepsis and as a marker to guide treatment strategies.
authors.affiliation: Department of Microbiology, Immunology and Parasitology, Medical Sciences Faculty, State University of Rio de Janeiro, 20 551-030, Rio de Janeiro, Brazil.
authors.forename: Gloria-Beatriz
authors.initials: GB
authors.lastname: Machado
authors.forename: Albanita V
authors.initials: AV
authors.lastname: de Oliveira
authors.forename: Alessandra M
authors.initials: AM
authors.lastname: Saliba
authors.forename: Carolina D M
authors.initials: CD
authors.lastname: de Lima
authors.forename: José H R
authors.initials: JH
authors.lastname: Suassuna
authors.forename: Maria-Cristina
authors.initials: MC
authors.lastname: Plotkowski
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001777:Blood Coagulation; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D005260:Female; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000378:metabolism*; D018410:Pneumonia, Bacterial / Q000097:blood* / Q000235:genetics / Q000382:microbiology; D011552:Pseudomonas Infections / Q000097:blood* / Q000235:genetics / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism / Q000382:microbiology; D018805:Sepsis / Q000097:blood* / Q000235:genetics / Q000382:microbiology; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D017395:Plasminogen Activator Inhibitor 1; D010972:Platelet Activating Factor; D012333:RNA, Messenger; C051321:SERPINE1 protein, human; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D005337:Fibrin