Captura e ativação de plasminogênio por Pseudomonas aeruginosa

• Ano de defesa: 2002
• Autor(a) principal: Silva, Cecilia Maria Ferreira da
• Outros Autores: [e-mail]
• Orientador(a): Plotkowski, Maria Cristina Maciel
• Banca de defesa: Andrade, João Ramos da Costa , Suasssuna, José Hermogenes Rocco , Passolani, Maria Cristina Vidal
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade do Estado do Rio de Janeiro
• Programa de Pós-Graduação: Programa de Pós-Graduação em Microbiologia
• Departamento: Centro Biomédico::Faculdade de Ciências Médicas
• País: Brasil
• Palavras-chave em Português: Pseudomonas aeruginosa; Captura e ativação de plasminogênio; Ativadores bacterianos de plasminogênio; Metástase bacteriana
• Palavras-chave em Inglês: Pseudomonas aeruginosa; Plasminogen binding and activation; Bacterial plasminogen activators; Bacterial metastasis
• Área do conhecimento CNPq: CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS
• Link de acesso: http://www.bdtd.uerj.br/handle/1/18170

This study was carried out to assess the affinity for plasminogen (Plg) by ELISA of Pseudomonas aeruginosa laboratory and clinical strains isolated from blood of septicemic patients or from stools of asymptomatic carriers. The Plg binding to ELISA microplate coated wells with 14 different bacterial strains was significantly higher (p<0.001) than the binding to uncoated control wells. The Plg binding by blood strains did not differ from the binding by stool strains, suggesting that the expression of Plg receptors on P. aeruginosa surface is constitutive. No difference in the Plg binding was detected between the piliated PAK strain and the isogeneic nonpiliated mutant PAK/p-, suggesting that type IV pili is not a main adhesin accountig for Plg binding to P. aeruginosa cells. The inclusion of ε-aminocaproic acid in the assays reduced significantly the Plg binding to PAK and PAK/p- strains indicating that Plg binding to surface P. aeruginosa receptors is lysin dependent and suggesting a role for the kringle domains in the Plg capture by bacterial cells. By Western-immunoblotting assays, several P. aeruginosa low molecular weight outer membrane proteins (from 30 to 60 kDa) were seen to react with Plg, suggesting that P.aeruginosa Plg surface receptors are omptins.To ascertain whether bacteria-bound Plg could be activated to plasmin, bacteria-coated microplate wells were incubated successively with Plg and with the urokinase (u-PA) activator. The bacteria- associated proteolytic activity was assessed by the addition of a chromogenic plasmin substrate. In parallel, other bacteria-coated microplate wells were exposed only to Plg and to the chromogenic substrate, to assess the intrinsic capability of the bacteria to generate plasmin. Most strains were shown to activate Plg in plasmin in presence of u-PA. Two clinical strains, one from blood and the other from stools, exhibited a marked capability to generate plasmin in the absence of u-PA, suggesting the existence of an endogenous cell surface Plg activator. The results from preliminary studies carried out in the presence of EDTA suggest that this P. aeruginosa Plg activator is a bacteria-bound serine protease. Finally, the role of Plg binding and activation to plasmin in the bacterial proteolytic activity and invasiveness was assessed by inoculating fibrin gels prepared in Durhan tubes or in Millicell inserts with plasmin-coated and uncoated bacteria. The presence of surface-bound plasmin enhanced significantly the P. aeruginosa capacity to invade the fibrin gells, detected as early as 3h after the infection. Based on these results, we suggest that in vivo, the binding of Plg to P. aeruginosa surface may represent a mechanism that could enhance the bacterial metastasis through normal host tissue barriers, such as fibrin clots and basal membranes, in the course of invasive infectious processe.