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Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores

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.

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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.

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Acknowledgements

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

Author information

Authors and Affiliations

Authors

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.

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The authors declare no competing financial interests.

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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.

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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

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