Atividade pró-coagulante da toxina ExoU de Pseudomonas aeruginosa: efeito sobre a expressão do fator tissular em células epiteliais respiratórias

• Autor(a) principal: Feliciano, Luís Filipe Pereira
• Data de Publicação: 2008
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14417
• topic: Pseudomonas aeruginosa; ExoU; Tissue factor; Airway epithelial cells; Procoagulant activity; Pseudomonas aeruginosa; ExoU; Fator tissular; Células epiteliais aéreas; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA

To evaluate the capacity of the P. aeruginosa toxin ExoU to induce the expression of tissue factor (TF) by epithelial respiratory cells from the BEAS-2B cell line, cells infected with the ExoU-producing PA103 bacterial strain were compared with cells infected with a mutant obtained by deletion of the exoU gene and with control non-infected cells in their i) expression of the TF mRNA, by RT-PCR; ii) expression of the protein TF in cell lisates and surfaces, by enzyme immunoassay (ELISA) and flow cytometry, respectively; iii) procoagulant activity by determining the ability of intact cells to induce the coagulation of normal human plasma and the ability of cell lysates to cleave the synthetic substrate of a chromogenic assay; iv) presence of soluble TF in cell culture supernatants, by ELISA and v) release of microparticles simultaneously expressing TF and phosphatidylserine, by flow cytometry. Cells infected with the wild type bacteria exhibited increased expression of TF mRNA 1 hour after infection and a positive modulation of TF expression in both cell lysates and cell surfaces. The enhancement of TF expression was inhibited when cells were infected with bacteria previously treated with a PLA2 inhibitor (MAFP), confirming that the ability of ExoU to modulate TF expression depended on its phospholipase A2 activity. Newly expressed TF was shown to be functionally active, by both the decrease in the clotting time of human plasma and the enhancement of the hydrolysis of the chromogenic assay substrate. Cells infected with the ExoU-producing bacteria exhibited also higher concentrations of soluble TF and of TF and PS bearing microparticles in the cell culture supernatants. These in vitro results were validated by our finding of increase TF concentrations in the lung parenchyma of mice infected intratracheally with the ExoU producing-bacteria at 24 h post-infection.