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.

  • Letter
  • Published:

Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases

Abstract

Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death1,2,3. In mouse macrophages, activation of this pathway requires the production of type-I interferons4,5, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

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

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

Figure 1: Caspase-11 activation by intracellular bacterial pathogens requires GBPs.
Figure 2: GBPs control bacterial replication.
Figure 3: Autophagy reduces caspase-11 activation.
Figure 4: GBP-mediated lysis of the PCV releases Salmonella into the cytosol.

Similar content being viewed by others

References

  1. Kayagaki, N. et al. Non-canonical inflammasome activation targets caspase-11. Nature 479, 117–121 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246–1249 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. 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  PubMed  PubMed Central  Google Scholar 

  4. Broz, P. et al. Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1. Nature 490, 288–291 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Case, C. L. et al. Caspase-11 stimulates rapid flagellin-independent pyroptosis in response to Legionella pneumophila. Proc. Natl Acad. Sci. USA 110, 1851–1856 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Casson, C. N. et al. Caspase-11 activation in response to bacterial secretion systems that access the host cytosol. PLoS Pathog. 9, e1003400 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. MacMicking, J. D. Interferon-inducible effector mechanisms in cell-autonomous immunity. Nature Rev. Immunol. 12, 367–382 (2012)

    Article  CAS  Google Scholar 

  9. Shenoy, A. R. et al. GBP5 promotes NLRP3 inflammasome assembly and immunity in mammals. Science 336, 481–485 (2012)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Kim, B. H. et al. A family of IFN-gamma-inducible 65-kD GTPases protects against bacterial infection. Science 332, 717–721 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Yamamoto, M. et al. A cluster of interferon-gamma-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii. Immunity 37, 302–313 (2012)

    Article  CAS  PubMed  Google Scholar 

  12. Broz, P. et al. Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J. Exp. Med. 207, 1745–1755 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Degrandi, D. et al. Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication. Proc. Natl Acad. Sci. USA 110, 294–299 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  15. VanCott, J. L. et al. Regulation of host immune responses by modification of Salmonella virulence genes. Nature Med. 4, 1247–1252 (1998)

    Article  CAS  PubMed  Google Scholar 

  16. Burton, N. A. et al. Disparate impact of oxidative host defenses determines the fate of Salmonella during systemic infection in mice. Cell Host Microbe 15, 72–83 (2014)

    Article  CAS  PubMed  Google Scholar 

  17. Thurston, T. L., Wandel, M. P., von Muhlinen, N., Foeglein, A. & Randow, F. Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482, 414–418 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Deretic, V., Saitoh, T. & Akira, S. Autophagy in infection, inflammation and immunity. Nature Rev. Immunol. 13, 722–737 (2013)

    Article  CAS  Google Scholar 

  19. Paetzold, S., Lourido, S., Raupach, B. & Zychlinsky, A. Shigella flexneri phagosomal escape is independent of invasion. Infect. Immun. 75, 4826–4830 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hunn, J. P. et al. Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii. EMBO J. 27, 2495–2509 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Traver, M. K. et al. Immunity-related GTPase M (IRGM) proteins influence the localization of guanylate-binding protein 2 (GBP2) by modulating macroautophagy. J. Biol. Chem. 286, 30471–30480 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Haldar, A. K. et al. IRG and GBP host resistance factors target aberrant, “non-self” vacuoles characterized by the missing of “self” IRGM proteins. PLoS Pathog. 9, e1003414 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Coers, J. Self and non-self discrimination of intracellular membranes by the innate immune system. PLoS Pathog. 9, e1003538 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mariathasan, S. et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Zhao, Z. et al. Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens. Cell Host Microbe 4, 458–469 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lima-Junior, D. S. et al. Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania. Nature Med. 19, 909–915 (2013)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank N. Mizushima and S. Virgin for Atg5-deficient BMDMs, K. Pfeffer for Gbp2-deficient BMDMs, J. Frey for B. thailandensis, the Biozentrum Proteomics and Imaging Core Facilities for technical assistance, K. Anderson, T. Soukup, R. Schwingendorf, J. C. Cox, V. M. Dixit for reagents and N. Personnic for discussions. This work was supported by an SNSF Professorship PP00P3_139120/1, University of Basel project grant ID2153162 to P.B. and a Marie Heim-Voegtlin Fellowship 145516 to D.K.B.

Author information

Authors and Affiliations

Authors

Contributions

E.M. and P.B. designed the study and wrote the manuscript. E.M., R.F.D., M.S.D., N.S. and P.B. performed the experiments and analysed data; D.K.B., D.B., S.W., M.R.-G., N.K., M.Y. and K.T. contributed reagents.

Corresponding author

Correspondence to Petr Broz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Type-I-interferon signalling is required to induce caspase-11-dependent cell death in response to bacterial infection, but not in response to LPS transfection.

a, LDH release from unprimed BMDMs infected for 16 h with wild-type (WT) S. typhimurium or ΔSPI-2 S. typhimurium grown to stationary phase. b, LDH release from primed BMDMs transfected with LPS O111:B4. Graphs show the mean and s.d. of quadruplicate wells and are representative of three independent experiments.

Extended Data Figure 2 BMDMs from Gbpchr3 KO mice have normal responses to priming stimuli, but fail to activate the non-canonical inflammasome during bacterial infections.

a, Schematic representation of the GBP locus on murine chromosome 3. The extent of the deletion in Gbpchr3 KO mice is indicated. bd, Induction of pro-caspase-11, GBP2 and GBP5 expression in lysates of wild-type and Gbpchr3 KO BMDMs stimulated for 16 h with the indicated amounts of murine IFN-β, murine IFN-γ or LPS O111:B4. e, TNF-α release from BMDMs stimulated for 16 h with LPS O111:B4. f, g, LDH release and IL-1β secretion from wild-type and Gbpchr3 KO BMDMs infected for 16 h with wild-type (WT) S. typhimurium, ΔSPI-2 S. typhimurium, V. cholerae, E. cloacae or C. koseri grown to stationary phase. Cells were primed overnight with LPS (f) or poly(I:C) (g). *Indicates background band. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two independent experiments. **P < 0.01, NS, not significant (two-tailed t-test).

Extended Data Figure 3 GBPs assist the detection of bacteria that escape into the cytosol only in primed macrophages.

ac, LDH release, IL-1β secretion and immunoblots for processed caspase-1 and caspase-11 released from unprimed BMDMs infected for 8–16 h with ΔsifA S. typhimurium or B. thailandensis grown to stationary phase. d, LDH release and IL-1β secretion from unprimed or IFN-γ-primed BMDMs infected for 16 h with ΔsifA S. typhimurium grown to stationary phase. Ext, extract; SN, supernatant. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two independent experiments. *P < 0.05; **P < 0.01; NS, not significant (two-tailed t-test).

Extended Data Figure 4 Murine GBP2 controls non-canonical inflammasome activation during Salmonella infection, but is dispensable for direct LPS sensing and canonical inflammasomes.

a, Schematic drawing of the inflammasome pathways activated by flagellin-deficient Salmonella. bd, LDH release, IL-1β secretion and immunoblots for processed caspase-1 and processed IL-1β released from unprimed BMDMs infected for 17 h with Δflag S. typhimurium grown to stationary phase. BMDMs were treated with the indicated siRNA for 56 h before infection. e, Immunoblots for processed caspase-1, IL-18 and caspase-11 released from unprimed BMDMs infected for 16 h with ΔSPI-2 S. typhimurium, E. cloacae or C. koseri grown to stationary phase. f, g, LDH release and IL-1β secretion from primed wild-type and Gbp2−/− BMDMs transfected with the indicated types of LPS for 16 h, treated with nigericin for 1 h, infected with SPI-1 T3SS expressing logarithmic phase wild-type S. typhimurium for 1 h, or transfected with poly(dA:dT) for 6 h. Cell were primed with PAM3CSK4 in f or LPS g. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two (e) and three (bd, f, g) independent experiments. NT, non-targeting siRNA; GM, GenMute transfection reagent; NS, not significant (two-tailed t-test).

Extended Data Figure 5 Normal activation of non-canonical and canonical inflammasomes in Gbp5−/− BMDMs.

a, Expression of GBP5 in wild-type and two lines of Gbp5−/− BMDMs (1 and 2). *Indicates a cross-reactive band. be, LDH release and IL-1β secretion from BMDMs infected for 16 h with wild-type (WT) S. typhimurium, ΔSPI-2 S. typhimurium, V. cholerae, E. cloacae or C. koseri grown to stationary phase (b), transfected with the indicated LPS for 16 h (c) infected for 1 h with SPI-1 T3SS expressing logarithmic phase wild-type S. typhimurium (d), or treated with 5 mM ATP or 20 mM nigericin for 4 h (e). Cells were left unprimed (b) or primed with PAM3CSK4 in (c) or LPS (d, e). Graphs show the mean and s.d. of triplicate or quadruplicate wells and data are representative of three independent experiments.

Extended Data Figure 6 GBPs control bacterial replication.

c.f.u.s at 16 h post-infection in wild-type and Gbpchr3 KO BMDMs infected with the indicated bacterial strains. Experiments are representative of two independent experiments.

Extended Data Figure 7 Inhibition of ROS and NO production does not affect non-canonical inflammasome activation.

a, b, ROS levels, LDH release and IL-1β secretion in unprimed BMDMs left uninfected or infected for 16 h with wild-type S. typhimurium grown to stationary phase. ce, LDH release, IL-1β secretion, ROS levels and immunoblots for processed caspase-1 and caspase-11 released from unprimed BMDMs infected for 16 h with wild-type (WT) S. typhimurium or E. cloacae grown to stationary phase in the presence of the ROS inhibitor (apocynin) or a vehicle control (DMSO). f, g, LDH release, IL-1β secretion and immunoblots for processed caspase-1 and caspase-11 released from unprimed BMDMs infected for 16 h with wild-type S. typhimurium or E. cloacae grown to stationary phase in the presence of the iNOS inhibitor (l-NAME) or a vehicle control (DMSO). h, NO release from unprimed or IFN-γ-primed BMDMs infected for 16 h with S. typhimurium in presence of the iNOS inhibitor (l-NAME) or a vehicle control (DMSO). Ext, extract; SN, supernatant. Graphs show the mean and s.d. of quadruplicate wells and data are representative of two (ac, eg) and three (d, h) independent experiments. NS, not significant (two-tailed t-test).

Extended Data Figure 8 Colocalization of GBPs and autophagy proteins on intracellular bacteria.

a, Colocalization of LC3 with GBPs in unprimed wild-type BMDMs infected with E. cloacae or C. koseri for 4 h and stained for LC3, GBP2 and DNA. b, Colocalization of galectin-8 and NDP52 in unprimed wild-type BMDMs infected with wild-type S. typhimurium for 4 h and stained for galectin-8, NDP52 and DNA. c, Colocalization of p62 and LC3 in unprimed wild-type BMDMs infected with wild-type S. typhimurium for 4 h and stained for LC3, p62 and DNA. d, Quantification of p62 and LC3 co-staining in wild-type and Gbpchr3 KO BMDMs at 4 h post-infection with Salmonella. Arrowheads indicate region shown in insets. Scale bars, 1 μm (a) and 10 μm (b, c). Graph shows the mean and s.d. of triplicate counts and images and graph are representative of at least two independent experiments. NS, not significant (two-tailed t-test).

Extended Data Figure 9 Digitonin-based quantification of cytoplasmic bacteria.

a, Immunostaining for calnexin and PDI (protein disulphide isomerase) in wild-type BMDMs left untreated or permeabilized with digitonin or saponin. b, Differentially permeabilized macrophages stained for cytosolic and vacuolar Salmonella at 4 h post-infection. c, Schematic representation of FACS-based analysis of cytosolic and vacuolar bacterial populations of Salmonella. Scale bars, 10 μm.

Extended Data Figure 10 Model for the role of GBPs and autophagy in caspase-11 activation.

The pathogen-containing vacuole of vacuolar bacterial pathogens is recognized by interferon-induced GBPs in an unknown manner. GBPs promote the lysis of the PCV either directly or indirectly, resulting in the release of the bacteria into the cytosol and activation of caspase-11 by bacterial LPS. β-galactosides of the lysed vacuole serve as danger signals upon exposure to the cytosol and are recognized by galectin-8 leading to the recruitment of the autophagy machinery. p62 participates in this process by recognizing ubiquitin-chains on the vacuole or the bacterium. Uptake of the bacterium and the lysed vacuole into autophagosomes reduces caspase-11 activation by removing the source of LPS from the cytosol.

Supplementary information

Supplementary Information

This file contains the sequences of siRNA pools used in this study. (PDF 95 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Meunier, E., Dick, M., Dreier, R. et al. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases. Nature 509, 366–370 (2014). https://doi.org/10.1038/nature13157

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13157

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

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