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The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus

Abstract

Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death1,2. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component3,4,5. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages6, is a universal component of the flagellin–NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5–NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity.

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Figure 1: A defined biochemical assay reveals a universal role of NAIP5 in flagellin-triggered NLRC4 inflammasome activation in mouse macrophages.
Figure 2: Flagellin interacts specifically with NAIP5 and the interaction correlates with the activity of flagellins from different bacteria.
Figure 3: Flagellin stimulates the NAIP5–NLRC4 association and reconstitution of flagellin activation of the NLRC4 inflammasome in non-macrophage cells.
Figure 4: NAIP2 interacts with the TTSS rod protein and is required for the rod protein to trigger mouse NLRC4 inflammasome activation.
Figure 5: C. violaceum infection studies reveal that the human NLRC4 inflammasome responds to the TTSS needle subunit through specific recognition by human NAIP.

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Acknowledgements

We thank V. Dixit for providing Nlrc4 and Asc knockout mice, K. Fitzgerald, D. Radzioch and A. Ding for immortalized macrophages, R. Vance for Naip5A/J cDNA, M. Donnenberg and J. Girón for EPEC strains, E. Miao for flagellin-deficient S. typhimurium strain, D. Milton and T. Hoang for bacterial vectors and T. Miki for C. violaceum strains. We are grateful to C. Yao for helping with flow cytometry, and Y. Xu and the NIBS animal facility for handling mouse lines. We thank members of the F.S. laboratory for helpful discussions and technical assistance. This work was supported by the National Basic Research Program of China (973 Programs, 2010CB835400 and 2012CB518700).

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Y.Z. and J.Y. performed experiments, assisted by J.S., Y.-N.G., Q.L., H.X. and L.L. Y.Z., J.Y. and F.S. analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Feng Shao.

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Zhao, Y., Yang, J., Shi, J. et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477, 596–600 (2011). https://doi.org/10.1038/nature10510

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