Atividade pró-trombótica da toxina ExoU de Pseudomonas aeruginosa detectada em modelos experimentais in vivo e in vitro

• Autor(a) principal: Machado, Glória Beatriz da Silva
• Data de Publicação: 2011
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14349
• topic: ExoU; Platelet activating factor (PAF); Plasminogen activator inhibitor type I (PAI-1); Pseudomonas aeruginosa; Sepsis; ExoU; Fator de Ativação plaquetária (PAF); Inibidor do ativador do plasminogênio tipo-1 (PAI-1); Pseudomonas aeruginosa; Sepse; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA::MICROBIOLOGIA MEDICA

Pseudomonas aeruginosa is an important agent of pneumonia, mainly in patients undergoing mechanical ventilation, which can progress to sepsis with high mortality rates. In sepsis, the systemic inflammatory process favors exacerbated imbalance between the coagulation and fibrinolysis pathways and the installation of a procoagulant state, leading to microvascular thrombosis, disseminated intravascular coagulation and multiple organ failure. Knowing the powerful proinflammatory activity of the P. aeruginosa toxin ExoU, secondary to its phospholipase A2 activity, the goal of this study was to investigate the ExoU potential to induce hemostatic changes related to sepsis pathogenesis. By using a murine model of pneumosepsis, obtained by the intratracheal injection of suspensions of the ExoU-producing PA103 P. aeruginosa strain or of its isogenic mutant PA103ΔexoU, defective in the toxin synthesis, ExoU was shown to enhance the severity of the infection and to induce higher mice mortality rate as well as leukopenia, thrombocytosis, vascular hyperpermeability and plasma transudation, evidenced, respectively, by the higher protein concentration in the bronchoalveolar lavage fluids (BALF) and accumulation of Evans blue dye, previously intravenous injectioned, in mice renal parenchyma. ExoU also favored platelet activation, evidenced by the higher concentration of platelets expressing P-selectin on their surface, greater number of platelet-derived microparticles and increased plasma concentration of thromboxane A2. Histopathology of the lungs and kidneys of PA103-infected animals confirmed the formation of microthrombi, which were not detected in controls or in animals infected with the bacterial mutant. In lungs, ExoU induced an intense inflammatory response with high concentrations of total and polymorphonuclear leukocytes, interleukin-6 and tumor necrosis factor-α in mice BALF. Immunohistochemical analysis showed intense fibrin deposition in the alveoli and interalveolar septa. The tissue factor-dependent procoagulant activity detected in BALF from PA103-infected mice did not depend on decreased production of tissue factor pathway inhibitor (TFPI) production, but was associated with increased concentration of plasminogen activator inhibitor-1 (PAI-1). To investigate the role of platelet activating factor (PAF) in PAI-1 release, we compared the activity of the enzyme PAF-acetylhydrolase (PAF-AH) in BALF from control and infected mice, and we observed that PAF-AH activity was significantly elevated in BALF from PA103-infected animals. Mice treatment with a PAF inhibitor prior to infection resulted in a significant reduction of PAI-1 concentration, as well as of BALF total leukocytes and procoagulant activity. In vitro, ExoU induced higher expression of PAI-1 m RNA and increased release of PAI-1 protein in supernatants from A549 epithelial respiratory cell cultures. A549 cell treatment with an anti-PAF receptor prior to infection significantly reduced PAI-1 concentration in supernatants from cells infected with the wild type strain. These results show a novel mechanism by which a baterial product can favor a prothrombotic activity and ExoU production by infecting P. aeruginosa isolates may have prognostic implications for patients.