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      Innate immune recognition of bacterial ligands by NAIPs dictates inflammasome specificity

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      Nature

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          Abstract

          Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the CASPASE1 (CASP1) protease 1, 2 . Although genetic data support a critical role for inflammasomes in immune defense and inflammatory diseases 3 , the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing C4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin 4, 5 and PrgJ 6 , a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homolog of NLRC4, NAIP5 (NLR family, Apoptosis Inhibitory Protein 5), has been implicated in activation of NLRC4 711 , but is widely assumed to play only an auxiliary role 1, 2 , since NAIP5 is often dispensable for NLRC4 activation 7, 8 . However, Naip5 is a member of a small multigene family 12 , raising the possibility of redundancy and functional specialization among Naip genes. Indeed, we show here that different NAIP paralogs dictate the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that NAIP2/NLRC4 physically associates with PrgJ but not flagellin, whereas NAIP5/NLRC4 associates with flagellin but not PrgJ. Taken together, our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP/NLRC4 inflammasomes.

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          Most cited references 15

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          Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf.

          Macrophages respond to Salmonella typhimurium infection via Ipaf, a NACHT-leucine-rich repeat family member that activates caspase-1 and secretion of interleukin 1beta. However, the specific microbial salmonella-derived agonist responsible for activating Ipaf is unknown. We show here that cytosolic bacterial flagellin activated caspase-1 through Ipaf but was independent of Toll-like receptor 5, a known flagellin sensor. Stimulation of the Ipaf pathway in macrophages after infection required a functional salmonella pathogenicity island 1 type III secretion system but not the flagellar type III secretion system; furthermore, Ipaf activation could be recapitulated by the introduction of purified flagellin directly into the cytoplasm. These observations raise the possibility that the salmonella pathogenicity island 1 type III secretion system cannot completely exclude 'promiscuous' secretion of flagellin and that the host capitalizes on this 'error' by activating a potent host-defense pathway.
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            Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.

            Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.
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              Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin.

              Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                29 July 2011
                28 August 2011
                29 March 2012
                : 477
                : 7366
                : 592-595
                Affiliations
                [1 ]Division of Immunology & Pathogenesis, Department of Molecular & Cell Biology, University of California, Berkeley, CA, USA 94720
                Author notes
                Correspondence and requests for materials should be addressed to R.E.V. ( rvance@ 123456berkeley.edu )
                [2]

                Contact: Russell E. Vance. 415 Life Science Addition, University of California, Berkeley, CA, USA 94720. rvance@ 123456berkeley.edu . Tel. (510) 643-2795.

                Article
                nihpa314562
                10.1038/nature10394
                3184209
                21874021

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                Funding
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: R01 AI080749-03 || AI
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: R01 AI075039-04 || AI
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