Efeito do Sistema de Secreção do Tipo VI de <i>Pseudomonas aeruginosa</i> em modelo murino de pneumonia aguda

• Ano de defesa: 2015
• Autor(a) principal: Santos, Sabrina Alves Oliveira Lima
• Orientador(a): Saliba, Alessandra Mattos
• Banca de defesa: Assis, Maria Cristina de , Freitas-almeida, Angela Corrêa de , Manfro, Wânia Ferraz Pereira
• 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: BR
• Palavras-chave em Português: Sistemas de secreção bacterianos; Pseudomonas aeruginosa; Citocinas; Inflamação; Neutrófilos .; Sistema de Secreção do Tipo VI; Neutrófilos
• Palavras-chave em Inglês: Type VI Secretion System; Pseudomonas aeruginosa; Cytokines; Inflammation; Neutrophils
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA
• Link de acesso: http://www.bdtd.uerj.br/handle/1/14439

The Type VI Secretion System (T6SS), the latest secretion machinery described in Gram-negative bacteria, is evenly distributed among the various species of this group of microorganisms. This secretion apparatus is capable of injecting effector protein in target eukaryotic and prokaryotic cells. Studies on the role of the T6SS on microbial virulence revealed that this secretory system actively participates in the establishment of infection and contributes to the survival of bacteria within phagocytic cells. The genome of the <i>Pseudomonas aeruginosa</i> PAO1 strain has three loci encoding apparatus of T6SS, called H1-T6SS, H2-T6SS and H3-T6SS. However, little is known about the participation of the T6SS in the pathogenesis of <i>P. aeruginosa</i> infections. Thus, the present study investigated the role of H1-T6SS, H2-T6SS and H3-T6SS during acute lung infections. To this end, C57/BL6 mice were infected with different doses of wild type PAO1 or mutants strains PAO1ΔH1, PAO1ΔH2, PAO1ΔH3 or PAO1ΔH1ΔH2ΔH3. After 24 hours, bronchoalveolar lavages (BALs) were collected for total leukocyte and polymorphonuclear cell counts, and for ELISA quantification of KC, the chemokine for neutrophils, and the proinflammatory cytokines IL-1β and TNF-α. In other experiments, lungs, livers, spleens and kidneys were macerated to determine the bacterial load and the occurrence of systemic dissemination of bacteria. The cytotoxicity of the T6SS on human neutrophils was determined <i>in vitro</i> by cell staining with propidium iodide (PI) and annexin-V followed by flow cytometry analysis. The results showed that the inactivation of the three T6SS significantly reduced the concentration of neutrophils in mice BALs when the animals were infected with 10<SUP>7</SUP> <i>P. aeruginosa</i> colony forming units. At this dose, it was observed that the median number of bacteria detected in animals infected with the T6SS mutants were lower than the numbers detected in animals infected with the parental PAO1 strain. The mutations in T6SS did not affect the systemic dissemination of bacteria. The investigation of the levels of proinflammatory cytokines showed a reduction in the median of TNF-α concentration in BALs from mice infected with the triple mutant PAO1ΔH1ΔH2ΔH3, compared to the median of the concentration in BALs from mice infected with the parental strain, although this difference was not statistically significant. Since the investigation of IL-1β and KC levels did not contribute to the elucidation of the mechanisms accounting for the reduction of neutrophil concentrations in BALs of mice infected by the triple PAO1 mutant, it was next investigated the possible role of the T6SS in the death of human neutrophils. No significant differences were detected when the different samples of infected cells were compared with each other. In conclusion, the results from this present study showed that the T6SS likely interferes in neutrophil response during mice acute pneumonia, but additional studies are needed to determine the role of this secretion mechanism in the pathogenesis of <i>P. aeruginosa</i> infections.