Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores

Nature volume 535, pages 153–158 (2016)Cite this article

Subjects

Abstract

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: GSDMD-NT forms oligomers, disrupted by mutation of four positively charged residues.
Figure 2: GSDMD-NT localizes to the plasma membrane.
Figure 3: N-terminal gasdermin D binds to phosphatidyl serine and cardiolipin and forms pores on liposomes.
Figure 4: GSDMD-NT kills bacteria.

Similar content being viewed by others

References

  1. Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015)

    Article  ADS  CAS  Google Scholar 

  2. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015)

    Article  ADS  CAS  Google Scholar 

  3. Rathinam, V. A. et al. TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria. Cell 150, 606–619 (2012)

    Article  CAS  Google Scholar 

  4. 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)

    Article  ADS  CAS  Google Scholar 

  5. Law, R. H. et al. The structural basis for membrane binding and pore formation by lymphocyte perforin. Nature 468, 447–451 (2010)

    Article  ADS  CAS  Google Scholar 

  6. Montal, M. Design of molecular function: channels of communication. Annu. Rev. Biophys. Biomol. Struct. 24, 31–57 (1995)

    Article  CAS  Google Scholar 

  7. Rosado, C. J. et al. The MACPF/CDC family of pore-forming toxins. Cell. Microbiol. 10, 1765–1774 (2008)

    Article  CAS  Google Scholar 

  8. 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)

    CAS  PubMed  Google Scholar 

  9. Bordier, C. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604–1607 (1981)

    Article  CAS  Google Scholar 

  10. Dotiwala, F. et al. Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites. Nat. Med. 22, 210–216 (2016)

    Article  CAS  Google Scholar 

  11. Walch, M. et al. Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell 157, 1309–1323 (2014)

    Article  CAS  Google Scholar 

  12. 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)

    Article  CAS  Google Scholar 

  13. Leventis, P. A. & Grinstein, S. The distribution and function of phosphatidylserine in cellular membranes. Annu. Rev. Biophys. 39, 407–427 (2010)

    Article  CAS  Google Scholar 

  14. Schlame, M. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes. J. Lipid Res. 49, 1607–1620 (2008)

    Article  CAS  Google Scholar 

  15. Pasparakis, M. & Vandenabeele, P. Necroptosis and its role in inflammation. Nature 517, 311–320 (2015)

    Article  ADS  CAS  Google Scholar 

  16. Dondelinger, Y. et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Reports 7, 971–981 (2014)

    Article  CAS  Google Scholar 

  17. 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)

    Article  CAS  Google Scholar 

  18. Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012)

    Article  CAS  Google Scholar 

  19. Wilschut, J. & Papahadjopoulos, D. Ca2+-induced fusion of phospholipid vesicles monitored by mixing of aqueous contents. Nature 281, 690–692 (1979)

    Article  ADS  CAS  Google Scholar 

  20. He, W. T. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res. 25, 1285–1298 (2015)

    Article  CAS  Google Scholar 

  21. Finzel, B. C. et al. Crystal structure of recombinant human interleukin-1β at 2.0 Å resolution. J. Mol. Biol. 209, 779–791 (1989)

    Article  CAS  Google Scholar 

  22. Lamkanfi, M. & Dixit, V. M. Mechanisms and functions of inflammasomes. Cell 157, 1013–1022 (2014)

    Article  CAS  Google Scholar 

  23. Rühl, S. & Broz, P. Caspase-11 activates a canonical NLRP3 inflammasome by promoting K+ efflux. Eur. J. Immunol. 45, 2927–2936 (2015)

    Article  Google Scholar 

  24. 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)

    Article  CAS  Google Scholar 

  25. 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)

    CAS  PubMed  Google Scholar 

  26. Aachoui, Y. et al. Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978 (2013)

    Article  ADS  CAS  Google Scholar 

  27. 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)

    Article  CAS  Google Scholar 

  28. 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)

    Article  CAS  Google Scholar 

  29. Miao, E. A. et al. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat. Immunol. 11, 1136–1142 (2010)

    Article  CAS  Google Scholar 

  30. 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)

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by US NIH grant R01AI123265 (J.L.).

Author information

Author notes

  1. Xing Liu, Zhibin Zhang and Jianbin Ruan: These authors contributed equally to this work.

Authors and Affiliations

  1. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, 02115, Massachusetts, USA

    Xing Liu, Zhibin Zhang, Jianbin Ruan, Venkat Giri Magupalli, Hao Wu & Judy Lieberman

  2. Department of Pediatrics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Xing Liu, Zhibin Zhang & Judy Lieberman

  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Jianbin Ruan, Venkat Giri Magupalli & Hao Wu

  4. Department of Dermatology and Harvard Skin Disease Research Center, Brigham and Women’s Hospital, Boston, 02115, Massachusetts, USA

    Youdong Pan

Authors
  1. Xing Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhibin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jianbin Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Youdong Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Venkat Giri Magupalli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hao Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Judy Lieberman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.L. conceived the study. X.L., Z.Z., J.R., H.W. and J.L. designed the experiments and analysed the data. Experiments were performed as follows (X.L., Figs 1a–d, 2, 3a-d, 4a, c-f, h Extended Data Fig. 1a–c, e, f; Z.Z. Figs 1e–g 3b, d, Fig, 4b, e–h, Extended Data Fig. 1d; J.R. Figs 3, 4e, f; Y.P. Fig. 2e, f; V.M. Fig. 3e). X.L., H.W. and J.L. wrote the manuscript.

Corresponding authors

Correspondence to Hao Wu or Judy Lieberman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information

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

Extended Data Figure 1 GSDMD-NT oligomerizes and induces pyroptosis.

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.

Supplementary information

Supplementary Information

This file contains the uncropped gel source data. (PDF 573 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature18629

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing