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Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms

Nature Immunology volume 21, pages 880–891 (2020)Cite this article

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Abstract

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP–caspase-4 pathway in antibacterial defense.

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Fig. 1: IFN-γ prevents proliferation of cytosol-invading S. Typhimurium.
Fig. 2: Cytosol-invading bacteria recruit CASP4 for GSDMD-dependent pyroptosis.
Fig. 3: GBPs recruit and activate CASP4 at the surface of cytosol-invading bacteria.
Fig. 4: Formation of LPS-dependent GBP–CASP4 complexes.
Fig. 5: Analysis of LPS-dependent GBP–CASP4 complexes.
Fig. 6: Structure–function analysis of the GBP-dependent CASP4 signaling platform.
Fig. 7: Gbps and Casp11 protect mice epistatically against bacterial infection.
Fig. 8: Processing and secretion of IL-18 require GBP-dependent CASP4 activity.

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Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files). The source data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank J. Kendrick-Jones (MRC Laboratory of Molecular Biology, Cambridge) for providing antiserum against NDP52. This work was supported by the MRC (grant no. U105170648) and the Wellcome Trust (grant no. WT104752MA) to F.R., and by the NIH National Institutes of Allergy and Infectious Diseases (grant nos. R01AI068041-13 and R01AI108834-05) to J.D.M. J.D.M. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK

    Michal P. Wandel, Keith B. Boyle & Felix Randow

  2. Howard Hughes Medical Institute and Systems Biology Institute, Yale University, West Haven, CT, USA

    Bae-Hoon Kim, Eui-Soon Park & John D. MacMicking

  3. Departments of Immunobiology and Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

    Bae-Hoon Kim, Eui-Soon Park & John D. MacMicking

  4. Department of Paediatrics, University of Cambridge, Cambridge, UK

    Komal Nayak & Matthias Zilbauer

  5. Department of Paediatric Gastroenterology, Hepatology and Nutrition, Cambridge University Hospitals, Addenbrooke’s Hospital, Cambridge, UK

    Komal Nayak & Matthias Zilbauer

  6. CIRI, Centre International de Recherche en Infectiologie, University of Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, University of Lyon, Lyon, France

    Brice Lagrange & Thomas Henry

  7. Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada

    Adrian Herod & John Rohde

  8. Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK

    Felix Randow

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Contributions

M.P.W. performed and analyzed all experiments with the following exceptions: K.B.B. performed and analyzed experiments in enterocytes and bacterial proliferation assays in knockout cells; B.-H.K., E.-S.P. and J.D.M. designed, performed and analyzed Salmonella and Shigella infections in mice; A.H. and J.R. generated Shigella mutants; K.N. and M.Z. generated human enteroids; and B.L. and T.H. generated U937 knockouts. M.P.W. and F.R. designed the study and wrote the manuscript.

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Correspondence to Michal P. Wandel or Felix Randow.

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Extended data

Extended Data Fig. 1 IFN-γ prevents proliferation of cytosol-invading S. Typhimurium.

a, Lysates of HeLa cells treated with the indicated siRNAs. Blots were probed with the indicated antibodies, PCNA – loading control. b, Colony-forming units (CFU) of S. Typhimurium in HeLa cells at 1h p.i.. c, Fold replication of S. Typhimurium ΔprgH +inv in HeLa cells. d, Confocal micrographs of HeLa cells infected with S. Typhimurium taken at 1 h p.i. stained with DAPI and antibodies against Galectin-8 and ubiquitin (FK2 antibody) (top panel) or over-expressing GFP::LC3C and stained with DAPI and antibody against NDP52 (bottom panel). e, Percentage of Annexin V positive Hela cells expressing CFP::Galectin-8 amongst cells harbouring intracellular S. Typhimurium. Negative or positive – none or at least one bacterium per cell positive for CFP::Galectin-8. Live imaged every 6 min for 6 h, 12 fields per condition. f, Percentage of PI positive nuclei in HeLa cells infected with S. Typhimurium at 2h p.i.. Cells were treated with DMSO, 50 μM NEC-1s, 10 μM NSA or 50 μM Z-VAD-FMK as indicated. g, Confocal micrograph of HeLa cells infected with S. Typhimurium in the presence of FAM-VAD-FMK and stained with DAPI and antibody against Galectin-8. Image taken at 90 min p.i.. Statistical significance was assessed by two-tailed unpaired Student’s t-test (b), one-way (e,f) or two-way (c) analysis of variance (ANOVA) with Tukey’s multiple comparisons test; ns, not significant, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (c, e, f) or five (b) independent experiments, or representative of two (a) or three (d, g) independent experiments. HeLa cells were treated with IFN-γ (g) or treated with IFN-γ as indicated (a-c, e, f). Bacteria were counted based on their ability to grow on agar plates (b, c). Scale bar, 10 μm (d, g). Uncropped blots (a) are shown in the Source Data. PI - propidium iodide, p.i. - post-infection, S.T. - S. Typhimurium.

Source data

Extended Data Fig. 2 Cytosol-invading bacteria recruit caspase-4.

a, Confocal micrographs of HeLa cells over-expressing GFP::Caspase-4 or -5 at 1 h p.i. with S. Typhimurium and stained with DAPI. Scale bar, 10 μm. b, f, g, Lysates of HeLa cells expressing the indicated GFP::Caspase constructs (b), of cells treated with the indicated siRNAs (f), or of the indicated control or knock-out cells (g). Blots were probed with indicated antibodies, PCNA (b, f), Actin (g) – loading control. Samples in Extended Data Fig. 2g, Fig. 2p and Fig. 8c were obtained from the same experiment. c, d, Percentage of S. Typhimurium positive for the indicated GFP::Caspase constructs (c) or staining positive for endogenous Galectin-8 and/or Caspase-4 (d) in HeLa cells at 1 h p.i.. n > 100 bacteria per coverslip, in triplicate. e, Percentage of FAM-VAD-FMK positive S. Typhimurium amongst bacteria staining positive for endogenous Galectin-8 at 90 min p.i. in HeLa cells treated with siRNAs against caspases as indicated. n > 100 Galectin-8 +ve bacteria per coverslip, in triplicate. h, Percentage of PI positive nuclei in the indicated control or knock-out HeLa cells uninfected or infected with S. Typhimurium at 2h p.i.. i, Fold replication of S. Typhimurium in HeLa cells treated with the indicated siRNAs against caspases. Bacteria were counted based on their ability to grow on agar plates. Statistical significance was assessed by one-way (e, h) or two-way (i) analysis of variance (ANOVA) with Tukey’s multiple comparisons test; ns, not significant, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (c, d, e, i) independent experiments, or representative of two (b, f, g) or three (a) independent experiments. HeLa cells were treated with IFN-γ (c) or treated with IFN-γ as indicated (a, d-i). Uncropped blots (b, f, g) are shown in the Source Data. PI - propidium iodide, p.i. - post-infection, +ve – positive.

Source data

Extended Data Fig. 3 The S. flexneri effector OspC3 inhibits interferon-induced pyroptosis.

a, PI positive nuclei in HeLa cells infected with the indicated S. flexneri strains at 2h p.i.. b, Percentage of PI positive nuclei in CFP::Galectin-8 expressing HeLa cells infected with the indicated S. flexneri strains in the presence of PI. n = three (WT) and four (ΔospC3) independent repeats. Live imaged every 5 min for 5 h, 10 fields per condition. c, Percentage of Zombie Green positive (that is dead) cells in monolayers of differentiated human epithelial organoids at 2 h p.i. with the indicated S. flexneri strains. n > 50 infected cells per coverslip. d, Confocal micrograph of a monolayer of differentiated human epithelial organoids stained with Zombie Green and antibody against ZO-1 at 1 h p.i. with the indicated S. flexneri strains. Scale bar, 10 μm. e, f, Fold replication of the indicated S. flexneri strains in HeLa cells. Bacteria were counted based on their ability to grow on agar plates. Statistical significance was assessed by one-way (a, b) or two-way (e, f) analysis of variance (ANOVA) with Tukey’s multiple comparisons test; ns, not significant, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (a, b, e, f) or four (b) independent experiments, or representative of two (d) independent experiments. HeLa cells were treated with IFN-γ as indicated (a-f). PI - propidium iodide, p.i. - post-infection.

Extended Data Fig. 4 Cytosol-invading bacteria trigger caspase-4 and gasdermin-D dependent pyroptosis.

a, b, g, Fold replication of S. flexneri ΔospC3 in HeLa cells treated with the indicated siRNAs against caspases (a) and in control or knock-out HeLa cells (b, g). Bacteria were counted based on their ability to grow on agar plates. c, d, Lysates of HeLa cells at 1 h p.i. with the indicated S. flexneri strains. Pro - full length pro-form of GSDMD (shorter exposure), NT - N-terminal domain of GSDMD (longer exposure). Samples in Extended Data Figs. 4c,d and 9a, c were obtained from the same experiment. e, h, Lysates of HeLa cells treated with the indicated siRNAs. * unspecific band. f, PI positive nuclei in the indicated control or knock-out HeLa cells at 2 h p.i. with S. flexneri ΔospC3. Statistical significance was assessed by one-way (f) or two-way (a, b, g) analysis of variance (ANOVA) with Tukey’s multiple comparisons test; ns, not significant, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (b, g, f) or four (a) independent experiments, or representative of two (e, h) or three (c, d) independent experiments. HeLa cells were treated with IFN-γ as indicated (a-h). Blots were probed with indicated antibodies, PCNA (e, h), Actin (c, d) – loading control. Uncropped blots (c-e, h) are shown in the Source Data. PI - propidium iodide, p.i. - post-infection.

Source data

Extended Data Fig. 5 GBP1 recruits GBP2-4 to S. Typhimurium.

a, b, Lysates of HeLa cells treated with the indicated siRNAs (a), or from the indicated control or knock-out cells (b). Blots were probed with indicated antibodies, PCNA (a), Actin (b) – loading control. Samples in Extended Data Fig. 5b, 8c and 9f were obtained from the same experiment. c, Percentage of S. Typhimurium positive for the indicated GFP::GBP constructs at 1 h p.i. in the indicated control or knock-out HeLa cells. n > 100 bacteria per coverslip, in triplicate. d, Structured illumination micrograph of HeLa cells expressing GFP::GBP1 and antibody-stained for Galectin-8 at 1 h p.i. with S. Typhimurium. Scale bar, 1 μm. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (c); ns, not significant, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (c) independent experiments, or representative of two (a, b) or three (d) independent experiments. HeLa cells were treated with IFN-γ (c, d) or treated with IFN-γ as indicated (b). Uncropped blots (a, b) are shown in the Source Data. p.i. - post-infection.

Source data

Extended Data Fig. 6 GBPs target cytosol-invading S. Typhimurium.

Confocal micrographs of HeLa cells over-expressing GFP::GBP1-7 and stained with DAPI and antibody against NDP52 at 1 h p.i. with S. Typhimurium. Representative of three independent experiments. Scale bar, 10 μm.

Extended Data Fig. 7 GBPs recruit and activate caspase-4.

a, Percentage of S. Typhimurium positive for GFP::GBP1-4 and/or staining positive for endogenous Caspase-4 in HeLa cells at 1 h p.i.. n > 100 bacteria per coverslip, in triplicate. b, e, Percentage of endogenous Caspase-4 (b) or FAM-VAD-FMK (e) positive S. Typhimurium amongst bacteria staining positive for endogenous Galectin-8 in the indicated control or knock-out HeLa cells at 1h (b) or 90 min (e) p.i.. n > 100 Galectin-8 +ve bacteria per coverslip, in triplicate. c, f Percentage of endogenous Caspase-4 (c) or FAM-VAD-FMK (f) positive bacteria of the indicated S. flexneri strains in HeLa cells at 1 h p.i.. n > 100 bacteria per coverslip, in triplicate. d, Confocal micrograph of a monolayer of differentiated human epithelial organoids antibody-stained for GBP1 and Caspase-4 at 1 h p.i. with S. flexneri ΔipaH9.8. g, Confocal micrographs of HeLa cells treated with DMSO or Carfilzomib as indicated at 1h p.i. with S. flexneri ΔospC3 in the presence of FAM-VAD-FMK and stained with DAPI. h-j, Percentage of FAM-VAD-FMK positive S. flexneri ΔospC3 ΔipaH9.8 (h, j) or S. flexneri ΔospC3 (i) at 1 h p.i. in the indicated control or knock-out HeLa cells (h,j) or in HeLa cells treated with the indicated siRNAs against caspases and 1 μM Carfilzomib (i). n > 100 bacteria per coverslip, in triplicate. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (b, c, e, f, h-j); ns, not significant, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (a-c, e, f, h-j) independent experiments, or representative of two (d, g) independent experiments. HeLa cells were treated with IFN-γ (a, b, d, e, g, h) or treated with IFN-γ as indicated (c, f, i, j). Cells were treated with DMSO or 1 μM Carfilzomib as indicated (c,f, g). Scale bar, 10 μm (d, g). p.i. - post-infection, +ve – positive.

Extended Data Fig. 8 GBPs govern gasdermin-D dependent pyroptosis.

a, b, Percentage of FAM-VAD-FMK positive cells among HeLa cells containing S. Typhimurium positive for endogenous Galectin-8 at 90 min p.i. (a) or containing S. flexneri ΔospC3 at 1 h p.i. (b); cells treated with siRNAs against GBPs as indicated. n > 100 cells with Galectin-8 +ve bacteria (a) or n > 100 infected cells (b) per coverslip, in triplicate. c, Lysates of the indicated control or knock-out HeLa cells infected with S. flexneri ΔospC3 for 1h. Pro - full length pro-form of GSDMD (shorter exposure), NT - N-terminal domain of GSDMD (longer exposure). Samples in Extended Data Fig. 8c, Extended Data Fig. 5b and Extended Data Fig. 9f were obtained from the same experiment. d, e, Percentage of PI positive nuclei in the indicated control or knock-out HeLa cells at 2 h p.i. with S. Typhimurium (e) or S. flexneri ΔospC3 (d). f, Lysates of the indicated control or knock-out U937 cells. g, Sanger sequencing chromatogram of control and GBP3 knock-out U937 cells. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (a, b, d, e); ns, not significant, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (b, d, e) or four (a) independent experiments, or representative of two (c, f) independent experiments. HeLa cells were treated with IFN-γ as indicated (a-f). Blots were probed with indicated antibodies, Actin – loading control (c, f). Uncropped blots (c, f) are shown in the Source Data. PI - propidium iodide, p.i. - post-infection, +ve – positive. S. T - S. Typhimurium.

Source data

Extended Data Fig. 9 Processing and secretion of IL-18 during S. flexneri infection.

a, c, Lysates of HeLa cells prepared at 1 h p.i. with the indicated S. flexneri strains. Samples in Extended Data Fig. 9a, c and Extended Data Fig. 4c, d were obtained from the same experiment. b, d, Release of IL-18 from HeLa cells infected with the indicated S. flexneri strains for 1h. e, Lysates of HeLa cells expressing the indicated FLAG-tagged caspase alleles and treated with the indicated siRNAs prepared at 1 h p.i. with S. flexneri ΔospC3. f, Lysates of the indicated control or knock-out HeLa cells prepared at 1 h p.i. with S. flexneri ΔospC3. Samples in Extended Data Fig. 9f, Extended Data Fig. 5b and Extended Data Fig. 8c and were obtained from the same experiment. g, Release of IL-18 from the indicated control or knock-out HeLa cells infected with S. flexneri ΔospC3 for 1h. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (b, d, g); ns, not significant, *P < 0.05, **P < 0.01 (exact p values are provided in Supplementary Table 1). Data are expressed as the Mean ± SEM of three (b, d, g) independent experiments, or representative of two (e, f) or three (a, c) independent experiments. HeLa cells were treated with IFN-γ as indicated (a-g). Blots were probed with indicated antibodies, Actin – loading control (a, c, e, f). Uncropped blots (a, c, e, f) are shown in the Source Data.

Source Data

Extended Data Fig. 10 Schematic illustration of the GBP-CASP4 pathway.

Interferon-induced guanylate-binding proteins (GBPs) transform Gram-negative bacteria into a caspase activation platform by coating their surface with a polyvalent protein array. The bacterial GBP coat may serve to foster contacts between CASP4 and its microbial ligand, the hydrophobic lipid A moiety of LPS, an integral and otherwise inaccessible component of the bacterial outer membrane. GBPs control CASP4 activation in a hierarchical manner; GBP1 initiates platform assembly, GBP2 and GBP4 control CASP4 recruitment, whereas GBP3 governs CASP4 activation. Once activated CASP4 cleaves GSDMD and IL-18 to cause pyroptotic cell death and cytokine release, thereby destroying the bacterial niche and alerting neighbouring cells of imminent danger. The cytosol-adapted bacterium Shigella flexneri antagonizes the pathway through secretion of the CASP4 inhibitor OspC3.

Supplementary information

Reporting Summary

Supplementary Table 1

Table containing precise P values.

Supplementary Video 1

Success and failure of galectin-8 to restrict proliferation of S. Typhimurium. Comparison of the outcome of cytosolic entry, marked by recruitment of galectin-8, for events 1 and 2. Live imaging on a confocal spinning disk microscope of HeLa cells expressing CFP::galectin-8 (white), infected with GFP-expressing S. Typhimurium (green) and live imaged every 6 min. Representative of six independent experiments. Scale bar, 10 μm.

Supplementary Video 2

Cytosolic entry of S. Typhimurium kills IFN-γ-treated cells. Illustration of the outcome of cytosolic entry, marked by recruitment of galectin-8, for two events. Live imaging on a confocal spinning disk microscope of IFN-γ-treated HeLa cells expressing CFP::galectin-8 (white), infected with GFP-expressing S. Typhimurium (green) in the presence of propidium iodide (red) and live imaged every 5 min. Representative of six independent experiments. Scale bar, 10 μm.

Supplementary Video 3

Caspase-4 recruitment to S. Typhimurium precedes cell death. Illustration of sequential events: (1) entry into the cytosol, marked by galectin-8; (2) recruitment of caspase-4; and (3) cell death, marked by nuclear accumulation of propidium iodide. Live imaging on a confocal spinning disk microscope of IFN-γ-treated HeLa cells expressing CFP::galectin-8 (white) and GFP::caspase-4 N104 (green), infected with mCherry-expressing S. Typhimurium (red) in the presence of propidium iodide (red) and live imaged every 5 min. Representative of three independent experiments. Scale bar, 10 μm.

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Wandel, M.P., Kim, BH., Park, ES. et al. Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat Immunol 21, 880–891 (2020). https://doi.org/10.1038/s41590-020-0697-2

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