title: Activation of NLRC4 by flagellated bacteria triggers caspase-1-dependent and -independent responses to restrict Legionella pneumophila replication in macrophages and in vivo.
abstract: Although NLRC4/IPAF activation by flagellin has been extensively investigated, the downstream signaling pathways and the mechanisms responsible for infection clearance remain unclear. In this study, we used mice deficient for the inflammasome components in addition to wild-type (WT) Legionella pneumophila or bacteria deficient for flagellin (flaA) or motility (fliI) to assess the pathways responsible for NLRC4-dependent growth restriction in vivo and ex vivo. By comparing infections with WT L. pneumophila, fliI, and flaA, we found that flagellin and motility are important for the colonization of the protozoan host Acanthamoeba castellanii. However, in macrophages and mammalian lungs, flagellin expression abrogated bacterial replication. The flagellin-mediated growth restriction was dependent on NLRC4, and although it was recently demonstrated that NLRC4 is able to recognize bacteria independent of flagellin, we found that the NLRC4-dependent restriction of L. pneumophila multiplication was fully dependent on flagellin. By examining infected caspase-1(-/-) mice and macrophages with flaA, fliI, and WT L. pneumophila, we could detect greater replication of flaA, which suggests that caspase-1 only partially accounted for flagellin-dependent growth restriction. Conversely, WT L. pneumophila multiplied better in macrophages and mice deficient for NLRC4 compared with that in macrophages and mice deficient for caspase-1, supporting the existence of a novel caspase-1-independent response downstream of NLRC4. This response operated early after macrophage infection and accounted for the restriction of bacterial replication within bacteria-containing vacuoles. Collectively, our data indicate that flagellin is required for NLRC4-dependent responses to L. pneumophila and that NLRC4 triggers caspase-1-dependent and -independent responses for bacterial growth restriction in macrophages and in vivo.
authors.affiliation: Department of Cell Biology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil.
authors.forename: Marcelo S F
authors.initials: MS
authors.lastname: Pereira
authors.forename: Giuliano F
authors.initials: GF
authors.lastname: Morgantetti
authors.forename: Liliana M
authors.initials: LM
authors.lastname: Massis
authors.forename: Catarina V
authors.initials: CV
authors.lastname: Horta
authors.forename: Juliana I
authors.initials: JI
authors.lastname: Hori
authors.forename: Dario S
authors.initials: DS
authors.lastname: Zamboni
mesh_terms: D048248:Acanthamoeba castellanii / Q000201:enzymology / Q000276:immunology / Q000382:microbiology*; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D058491:Bacterial Load / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics; D001854:Bone Marrow Cells / Q000201:enzymology / Q000276:immunology / Q000382:microbiology; D002135:Calcium-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D005260:Female; D005407:Flagella / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D005408:Flagellin / Q000096:biosynthesis / Q000235:genetics; D058847:Inflammasomes / Q000172:deficiency / Q000235:genetics; D016952:Legionella pneumophila / Q000235:genetics / Q000254:growth & development* / Q000276:immunology*; D008124:Locomotion / Q000276:immunology; D008264:Macrophages / Q000201:enzymology / Q000276:immunology* / Q000382:microbiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D006180:Proton-Translocating ATPases / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology
chemical_list: D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D058847:Inflammasomes; C512299:Ipaf protein, mouse; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; D005408:Flagellin; C067138:flaA protein, bacteria; C069251:fliI protein, bacteria; D006180:Proton-Translocating ATPases