Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β1,2. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015)
Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015)
Rathinam, V. A. et al. TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria. Cell 150, 606–619 (2012)
Hagar, J. A., Powell, D. A., Aachoui, Y., Ernst, R. K. & Miao, E. A. Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341, 1250–1253 (2013)
Law, R. H. et al. The structural basis for membrane binding and pore formation by lymphocyte perforin. Nature 468, 447–451 (2010)
Montal, M. Design of molecular function: channels of communication. Annu. Rev. Biophys. Biomol. Struct. 24, 31–57 (1995)
Rosado, C. J. et al. The MACPF/CDC family of pore-forming toxins. Cell. Microbiol. 10, 1765–1774 (2008)
Geourjon, C. & Deléage, G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. 11, 681–684 (1995)
Bordier, C. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604–1607 (1981)
Dotiwala, F. et al. Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites. Nat. Med. 22, 210–216 (2016)
Walch, M. et al. Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell 157, 1309–1323 (2014)
van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008)
Leventis, P. A. & Grinstein, S. The distribution and function of phosphatidylserine in cellular membranes. Annu. Rev. Biophys. 39, 407–427 (2010)
Schlame, M. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes. J. Lipid Res. 49, 1607–1620 (2008)
Pasparakis, M. & Vandenabeele, P. Necroptosis and its role in inflammation. Nature 517, 311–320 (2015)
Dondelinger, Y. et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Reports 7, 971–981 (2014)
Wang, H. et al. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol. Cell 54, 133–146 (2014)
Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012)
Wilschut, J. & Papahadjopoulos, D. Ca2+-induced fusion of phospholipid vesicles monitored by mixing of aqueous contents. Nature 281, 690–692 (1979)
He, W. T. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res. 25, 1285–1298 (2015)
Finzel, B. C. et al. Crystal structure of recombinant human interleukin-1β at 2.0 Å resolution. J. Mol. Biol. 209, 779–791 (1989)
Lamkanfi, M. & Dixit, V. M. Mechanisms and functions of inflammasomes. Cell 157, 1013–1022 (2014)
Rühl, S. & Broz, P. Caspase-11 activates a canonical NLRP3 inflammasome by promoting K+ efflux. Eur. J. Immunol. 45, 2927–2936 (2015)
Warren, S. E., Mao, D. P., Rodriguez, A. E., Miao, E. A. & Aderem, A. Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection. J. Immunol. 180, 7558–7564 (2008)
Cervantes, J., Nagata, T., Uchijima, M., Shibata, K. & Koide, Y. Intracytosolic Listeria monocytogenes induces cell death through caspase-1 activation in murine macrophages. Cell. Microbiol. 10, 41–52 (2008)
Aachoui, Y. et al. Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978 (2013)
Wu, J., Fernandes-Alnemri, T. & Alnemri, E. S. Involvement of the AIM2, NLRC4, and NLRP3 inflammasomes in caspase-1 activation by Listeria monocytogenes. J. Clin. Immunol. 30, 693–702 (2010)
Sauer, J. D. et al. Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol. Cell Host Microbe 7, 412–419 (2010)
Miao, E. A. et al. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat. Immunol. 11, 1136–1142 (2010)
Thiery, J., Walch, M., Jensen, D. K., Martinvalet, D. & Lieberman, J. Isolation of cytotoxic T cell and NK granules and purification of their effector proteins. Curr. Protoc. Cell Biol. 47, 3.37:3.37.1–3.37.29 (2010)
This work was supported by US NIH grant R01AI123265 (J.L.).
The authors declare no competing financial interests.
Nature thanks F. Sigworth and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, b, HEK293T cells, transfected with Flag–GSDMD-NT (a) or Flag–GSDMD (b), were lysed with or without N-ethylmaleimide or 2ME, and analysed by SDS–PAGE and Flag immunoblot. c, Lysates of HEK293T cells, transfected with HA–GSDMD-NT and/or Flag–GSDMD-NT, were immunoprecipitated with anti-HA and analysed by immunoblot with the indicated antibodies. d, HEK293T cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag and analysed by immunoblot with the indicated antibodies. Flag–GSDMD-NT (Flag-NT) was expressed at considerably lower levels than GSDMD-CT-MYC(CT-MYC) or Flag–GSDMD-CT (Flag-CT), which accounts for the relative weak intensity of the corresponding bands on the middle blot. e, HEK293T cells, transiently transfected with the indicated plasmids, were assessed 16 h after transfection for cell death by CytoTox96 assay. f, Immortalized iBMDMs expressing Flag–GSDMD were electroporated with PBS, ultra LPS or Pam3CSK4, as a negative control for pyroptosis. 2 h later, cell death was determined by CytoTox96 assay. Graphs show the mean ± s.d. of triplicate wells and data shown are representative of three independent experiments. **P < 0.01 (two-tailed t-test).
Extended Data Figure 2 Mutation of four positively charged residues in GSDMD-NT or of two cysteine residues disrupts pyroptosis.
a, Lysates of HEK293T cells, transfected with the indicated plasmids, were immunoprecipitated with anti-Flag and analysed by immunoblot with the indicated antibodies. The 4A mutant of GSDMD-NT does not self-associate in multimers. b, Mutations in other basic residues do not affect pyroptosis. The indicated wild-type or mutated Flag–GSDMD-NT constructs were transiently expressed in HEK293T cells. Medium was collected 18 h after transfection and cell death was measured by CytoTox96 assay. c, d, Knockdown in immortalized iBMDMs of Gsdmd and ectopic expression of wild-type or 4A Gsdmd mRNA (c, assessed by qRT–PCR relative to GAPDH) and protein (d, relative to tubulin). These data for the cells used in the rescue experiment in Fig. 1h show that the ectopic proteins are expressed at similar levels as the endogenous protein. e, Replacement of Cys37 or Cys192 by Ala in GSDMD-NT disrupts oligomerization. Mean ± s. d. of three technical replicates and data shown are representative of three independent experiments (b, c). Statistical differences are calculated by two-tailed t-test (in b, compared to samples transfected to express wild-type GSDMD-NT); **P < 0.01 (two-tailed t-test).
Extended Data Figure 3 Treatment with GSDMD-NT reduces bacterial viability, but does not affect the viability of mammalian cells.
a, Antibiotic-free culture supernatants (concentrated fivefold) from transfected HEK293T cells, collected 30 h after transfection, were added to iBMDMs, which were cultured at 37 °C in 200 μl final volume for 6 h before measuring viability by CellTiter-Glo. b, HEK293T cells, transfected with Flag–GSDMD-NT 6 h earlier, were mixed with an equal number of CFSE-labelled untransfected HEK293T cells and incubated for 18 h before assessing cell death by propidium iodide staining and flow cytometry. c, E. coli and S. aureus were untreated or treated with recombinant GSDMD, wild-type or 4A-mutant GSDMD-NT, or GSDMD-CT (200 nM or indicated concentrations) for 20 min before samples were collected and bacterial growth was assessed by monitoring turbidity by optical density (representative experiments, left). The time to reach OD600 of 0.05 above background, which is a quantitative measure of the lag in detectable growth because of fewer viable bacteria, was defined as Tthreshold (right). The right graph shows the mean ± s.d. of three technical replicates. d, Bacterial viability after 20 min incubation with indicated proteins (200 nM) or isopropanol. Syto-9 enters live and dead bacteria, PI only enters dead bacteria (representative images, left; percent live cells, right). e, Fluorescence microscopy of mCherry-expressing L. monocytogenes incubated with AlexaFluor 488-GSDMD (activated or not with caspase-11) or AlexaFluor488-GSDMD-CT for 30 min at 37 °C. Data shown are representative of results of three independent experiments. Statistical differences are relative to untreated samples; **P < 0.01 (two-tailed t-test). Scale bars, 5 μm.
About this article
Cite this article
Liu, X., Zhang, Z., Ruan, J. et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535, 153–158 (2016). https://doi.org/10.1038/nature18629
This article is cited by
Journal of Neuroinflammation (2023)
Homocysteine promotes atherosclerosis through macrophage pyroptosis via endoplasmic reticulum stress and calcium disorder
Molecular Medicine (2023)
Inhibition of TRAF6 improves hyperlipidemic acute pancreatitis by alleviating pyroptosis in vitro and in vivo rat models
Biology Direct (2023)
Lipocalin-2-mediated astrocyte pyroptosis promotes neuroinflammatory injury via NLRP3 inflammasome activation in cerebral ischemia/reperfusion injury
Journal of Neuroinflammation (2023)
Journal of Hematology & Oncology (2023)