ExoU, uma proteína do sistema de secreção do tipo III de Pseudomonas aeruginosa, induz a secreção de eicosanóides por células epiteliais respiratórias. Papel dos corpúsculos lipídicos neste processo

• Autor(a) principal: Brandão, Bruno Araújo
• Data de Publicação: 2006
• 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/1/14442
• topic: Pseudomonas aeruginosa; Secretion system type three; ExoU; Eicosanoids. Inflammatory response; Lipid Bodies; Pseudomonas aeuginosa; Sistema de secreção do tipo três; ExoU; Eicosanóides; Resposta Inflamatória; Corpúsculos lipídicos; Pseudomona aeruginosa; Células epiteliais; Secreção; Inflamação; Pulmões Infecções; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA

To ascertain the role of ExoU in airway inflammatory response, human bronchial epithelial cells from the BEAS-2B line were treated with radiolabeled AA and then infected with the ExoU-producing P. aeruginosa PA103 strain, and with the mutants PA103ΔexoU (with deletion of the gene exoU), PA103ΔUT/exoU (with deletion of exoU and complementation with the functional gene) and PA103ΔUT/S142A (with deletion of exoU and complementation with a gene with specific site mutagenesis in the enzyme catalytic domain). After 1h, the release of AA by cells infected with the ExoU-producing strains was significantly higher than the release by control cells and by cells infected with the ExoU deficient mutants. Bacterial treatment with MAFP, an inhibitor of PLA2, inhibited significantly the AA release. Cells infected with PA103 and PA103ΔUT/exoU secreted PGE2 and LTB4 in concentrations significantly higher than cells infected with the other bacterial strains and control non-infected cells. MAFP treatment inhibited significantly the release of PGE2. The flow cytometric analysis of infected and non-infected cells exposed to an anti-COX-2 antibody revealed that the percentage of PA103-infected cells labeled by the antibody was significantly higher than the percentage of labeled cells in control cultures but was similar to the percentage of labeled cells in cultures infected with the ExoU-deficient bacteria. Cell treatment with NS 398, a specific inhibitor of COX-2, resulted in a significant inhibition of PGE2 secretion by PA103-infected cells, but not by PA103ΔUT/exoU-infected cells or by control cells. Lipid bodies (LB) are cytoplasmic domains enriched in COX-2 and other enzymes implicated in the metabolismo of AA that can be induced by different stimuli, including bacterial LPS and AA. Since PA103-infected cells released high concentrations of free AA, we wondered whether the increased release of eicosanoids by these cells may have resulted from an AA-induced increase in LB numbers. To address this question, infected and non-infected cells were exposed to a lipophylic probe that labels LB. Flow cytometric showed that the percentage of labeled cells in cultures infected with the ExoU-producing bacteria was significantly lower than the percentage in control cultures or in cultures infected with the ExoU-deficient bacteria. MAFP treatment inhibited significantly the reduction of the labeling of PA103-infected cultures. Flow cytometric analysis of cultures exposed to the lypophylic probe and to an anti-PGE2 antibody showed, in PA103-infected cultures, the simultaneous reduction in cell labeling with the probe and the increase in the labeling with the anti-PGE2 antibody. Based in these results we speculate that in cultures infected with the ExoU-producing bacteria, the toxin may have metabolysed LB glycerophospholipids, leading both to a reduction in its affinity for the probe and to a local PGE2 synthesis.