RESUMO
Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Caspase 1/imunologia , Caspase 7/imunologia , Caspase 8/imunologia , Inflamassomos/imunologia , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Proteína Inibidora de Apoptose Neuronal/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Caspase 1/genética , Caspase 7/genética , Caspase 8/genética , Inflamassomos/genética , Peptídeos e Proteínas de Sinalização Intracelular , Doença dos Legionários/genética , Doença dos Legionários/patologia , Camundongos , Camundongos Knockout , Proteína Inibidora de Apoptose Neuronal/genética , Proteínas de Ligação a FosfatoRESUMO
Legionella pneumophila is an intracellular bacterium that was evolutionarily selected to survive in freshwater environments by infecting free-living unicellular protozoa. Once humans inhale contaminated water droplets, the bacteria reach the pulmonary alveoli where they are phagocytized by resident alveolar macrophages. Depending on host immunity and bacterial virulence genes, the infection may progress to an acute pneumonia called Legionnaires' disease, which can be fatal. Of note, an effective immune response is critical to the outcome of the human infection. These clinical observations highlight the importance of animal models of pulmonary infection for in vivo investigation of bacterial pathogenesis and host responses. In this chapter we provide detailed protocols for intranasal infection of mouse with L. pneumophila.