Envolvimento de peroxirredoxina LsfA na virulência de Pseudomonas aeruginosa [thesis]

Gilberto Hideo Kaihami
CNPq) pelo apoio financeiro. Aos meus pais Mario e Harumi que deram todo o apoio durante todas as fases da minha vida, com muito amor e dedicação. Aos meus irmãos Ricardo e Fabio, por fazerem parte da minha vida. À Dr a Regina Baldini pelo carinho, incentivo, dedicação neste trabalho, e mais que isso por acreditar que eu seria capaz de desenvolvê-lo. Ao Dr. Sandro de Almeida pelo incentivo, disponibilidade, e pela coragem ao aceitar desenvolver este trabalho conosco. À Dr a Suely Lopes Gomes
more » ... uely Lopes Gomes pelo apoio e disponibilidade do espaço físico, e pelos grandes conselhos e ensinamentos. Aos docentes que participaram da minha banca de qualificação, Dr. Ferreira e a Dr a Marilis do Valle Marques. Aos amigos do grupo da professora Regina: Ana Paula, Ana Laura, Eliezer, Patrícia, Thays e Duílio pelos ótimos momentos de descontração tornando o ambiente agradável para o trabalho. Gostaria de agradecer em especial ao Diogo e Gianlucca por me ensinarem as técnicas básicas de biologia molecular, além do carinho e atenção em todos os momentos desde a iniciação até o presente momento. Aos amigos do grupo do professor Sandro: Karla, José (Zé), Lucas, Fábio, Miriam, Pollyanna, Suelen, Lavinia e Grasi pela ajuda e pelo aprendizado e desenvolvimento pessoal e acadêmico. Ao grupo da professora Suely: Rogério, César, André, Gabriela, Juliana e Liliane pelo bom ambiente de trabalho. À Sandra, Luci, Doris e Nilde pela dedicação e competência ao trabalho. À Dona Maria e Dona Miriam pelo cafezinho de todo dia. Aos funcionários da secretaria de pós-graduação Cibele, Emiliano e Milton pela atenção e ajuda sempre que necessário. Aos amigos e colegas da Bioquímica e da Farmácia pelos sorrisos e conversas pelo corredor. Aos amigos Andreas, Mario (Moxila), Mauro (Boteco), Marião, Rapha, Thiago (Menudo), Luizinho, Akira, Deris, por acreditarem e apoiarem hoje e sempre. Palavras-chave: peroxirredoxina, macrófagos, pneumonia, Pseudomonas aeruginosa, virulência, imunidade inata ABSTRACT Kaihami, G.H. Involvement of peroxiredoxin LsfA in the virulence of Pseudomonas aeruginosa. Masters Thesis --Graduate Program in Biochemistry. Instituto de Química, Universidade de São Paulo, São Paulo. Bacteria are recognized by macrophages via Toll-Like Receptors (TLR), leading to a signaling pathway that activates NF-κB and MAPKs. Killing in phagossomes is achieved by reactive oxygen and nitrogen species (ROS/RNS) generation. P. aeruginosa is a common cause of ventilator associated pneumonia and it uses several strategies for virulence and defense, including antioxidant mechanisms. In this work, we show for the first time that the 1-Cys peroxiredoxin LsfA is implicated in P. aeruginosa virulence. Mutant strains with a deletion in lsfA or with a mutation (Cys45 to Ala, RB302) were constructed and they were more sensitive to H 2 O 2 than the wild type strain PA14, as verified by a growth inhibition assay. In vitro peroxidasic activity of LsfA was measured by ferric-thiocyanate assay, and while the wild-type protein was active, the mutation in Cys45 abolished its activity. Infection of J774 macrophages with ∆lsfA or C45A strains resulted in lower cell death, increased bacterial clearance and higher TNF-α production in comparison to PA14-infected macrophages, suggesting a higher level of MAPKs and NF-κB activation due to the mutant strains. To verify whether LsfA could modify the oxidative state of infected macrophages, they were infected with PA14 or RB302 strains and incubated with carboxy-H2DCFDA, an indicator that emits fluorescence when oxidized. Macrophages infected with mutant strains showed a higher oxidative state in comparison to PA14-infected cells, thus confirming that LsfA limits macrophages activation that leads to TNF-α production and cytotoxic activity. MAPKs and NF-kB pathways are required to full production of TNF-α in macrophages infected with RB302, as shown using pharmacological inhibitors for those pathways. When macrophages were infected with RB302 in the presence of the antioxidant N-acetylcysteine, there was a reduction in TNF-α production as compared to PA14, as expected. In an acute pneumonia model, all PA14-infected mice died at 48h postinfection, while C45A-infected mice survived as long as 60 days. There was also reduction in bacterial counts in the lungs, spleen and liver of mice infected with RB302, in comparison to PA14-infected mice. A greater pro-inflammatory cytokine production was observed in mice infected with mutant strain in comparison to mice infected with PA14. Altogether, this work shows for the first time the role of a bacterial 1-Cys Prx that modulates host immune response in vitro and in vivo.
doi:10.11606/d.46.2013.tde-15052013-091625 fatcat:4gvkuedpobfq5oybxsu24x3wkq