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Caspase-4 Activation and Recruitment to Intracellular Gram-Negative Bacteria

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Pyroptosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2641))

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Abstract

The non-canonical inflammasome pathway functions as the primary cytosolic innate immune detection mechanism for Gram-negative bacterial lipopolysaccharide (LPS) in human and mouse cells and controls the proteolytic activation of the cell death executor gasdermin D (GSDMD). The main effectors of this pathways are the inflammatory proteases caspase-11 in mice and caspase-4/caspase-5 in humans. These caspases have been shown to bind LPS directly; however, the interaction between LPS and caspase-4/caspase-11 requires a set of interferon (IFN)-inducible GTPases, known as guanylate-binding proteins (GBPs). These GBPs assemble to form coatomers on cytosolic Gram-negative bacteria, which function as recruitment and activation platforms for caspase-11/caspase-4. Here we describe an assay to monitor caspase-4 activation in human cells by immunoblotting and its recruitment to intracellular bacteria using the model pathogen Burkholderia thailandensis.

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References

  1. Schroder K, Tschopp J (2010) Review the Inflammasomes supplement. Cell 140:821. https://doi.org/10.1016/j.cell.2010.01.040

    Article  CAS  PubMed  Google Scholar 

  2. Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16:407. https://doi.org/10.1038/nri.2016.58

    Article  CAS  PubMed  Google Scholar 

  3. Shi J et al (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526:660. https://doi.org/10.1038/nature15514

    Article  CAS  PubMed  Google Scholar 

  4. Kayagaki N et al (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526:666. https://doi.org/10.1038/nature15541

    Article  CAS  PubMed  Google Scholar 

  5. Shi J et al (2014) Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514:187. https://doi.org/10.1038/nature13683

    Article  CAS  PubMed  Google Scholar 

  6. Tretina, K., Park, E. S., Maminska, A. & MacMicking, J. D. Interferon-induced guanylate-binding proteins: Guardians of host defense in health and disease. Journal of Experimental Medicine vol. 216:482 (2019)

    Google Scholar 

  7. Santos JC, Broz P (2018) Sensing of invading pathogens by GBPs: at the crossroads between cell-autonomous and innate immunity. J Leukoc Biol 104:729. https://doi.org/10.1002/JLB.4MR0118-038R

    Article  CAS  PubMed  Google Scholar 

  8. Kim BH et al (2016) Interferon-induced guanylate-binding proteins in inflammasome activation and host defense. Nat Immunol 17:481. https://doi.org/10.1038/ni.3440

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. MacMicking JD (2012) Interferon-inducible effector mechanisms in cell-autonomous immunity. Nat Rev Immunol 12:367. https://doi.org/10.1038/nri3210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Santos JC et al (2020) Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria. Nat Commun 11:3276. https://doi.org/10.1038/s41467-020-16889-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wandel MP et al (2020) Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat Immunol 21:880. https://doi.org/10.1038/s41590-020-0697-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dilucca M, Ramos S, Shkarina K, Santos JC, Broz P (2021) Guanylate-binding protein-dependent noncanonical inflammasome activation prevents burkholderia thailandensis-induced multinucleated giant cell formation. MBio 12:e0205421

    Article  PubMed  Google Scholar 

  13. Julien O, Wells JA (2017) Caspases and their substrates. Cell Death Differ 2017 24:1380–1389

    Article  CAS  Google Scholar 

  14. Thornberry NA, Lazebnik Y (1998) Caspases: Enemies within. Science 281:1312–1316

    Article  CAS  PubMed  Google Scholar 

  15. Scott AM, Saleh M (2006) The inflammatory caspases: guardians against infections and sepsis. Cell Death Differ 2007 14:23–31

    Article  Google Scholar 

  16. Van Opdenbosch N, Lamkanfi M (2019) Caspases in cell death, inflammation, and disease. Immunity 50:1352–1364

    Article  PubMed  PubMed Central  Google Scholar 

  17. French CT et al (2011) Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade. Proc Natl Acad Sci U S A 108:12095. https://doi.org/10.1073/pnas.1107183108

    Article  PubMed  PubMed Central  Google Scholar 

  18. Galyov EE, Brett PJ, Deshazer D (2010) Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis. Annu Rev Microbiol 64:495. https://doi.org/10.1146/annurev.micro.112408.134030

    Article  CAS  PubMed  Google Scholar 

  19. Wiersinga WJ, van der Poll T, White NJ, Day NP, Peacock SJ (2006) Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei. Nat Rev Microbiol 4:272. https://doi.org/10.1038/nrmicro1385

    Article  CAS  PubMed  Google Scholar 

  20. Wiersinga WJ et al (2018) Melioidosis. Nat Rev Dis Prim 4:17107. https://doi.org/10.1038/nrdp.2017.107

    Article  PubMed  Google Scholar 

  21. Stevens MP et al (2002) An Inv/mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol 46:649. https://doi.org/10.1046/j.1365-2958.2002.03190.x

    Article  CAS  PubMed  Google Scholar 

  22. Benanti EL, Nguyen CM, Welch MD (2015) Virulent burkholderia species mimic host actin polymerases to drive actin-based motility. Cell 161:348. https://doi.org/10.1016/j.cell.2015.02.044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Toesca IJ, French CT, Miller JF (2014) The type VI secretion system spike protein VgrG5 mediates membrane fusion during intercellular spread by pseudomallei group Burkholderia species. Infect Immun 82:1436. https://doi.org/10.1128/IAI.01367-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lennings J, West TE, Schwarz S (2019) The Burkholderia type VI secretion system 5: composition, regulation and role in virulence. Front Microbiol 9:3339. https://doi.org/10.3389/fmicb.2018.03339

    Article  PubMed  PubMed Central  Google Scholar 

  25. Aachoui Y et al (2013) Caspase-11 protects against bacteria that escape the vacuole. Science 339:975. https://doi.org/10.1126/science.1230751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ceballos-Olvera I, Sahoo M, Miller MA, del Barrio L, Re F (2011) Inflammasome-dependent pyroptosis and IL-18 protect against burkholderia pseudomallei lung infection while IL-1β is deleterious. PLoS Pathog 7:e1002452. https://doi.org/10.1371/journal.ppat.1002452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kovacs SB et al (2020) Neutrophil Caspase-11 is essential to defend against a cytosol-invasive bacterium. Cell Rep 32:107967. https://doi.org/10.1016/j.celrep.2020.107967

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Department of Immunobiology, University of Lausanne, Epalinges, Switzerland

    Marisa Dilucca & Petr Broz

Authors
  1. Marisa Dilucca
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  2. Petr Broz
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Corresponding author

Correspondence to Petr Broz .

Editor information

Editors and Affiliations

  1. Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA

    Susan L. Fink

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Dilucca, M., Broz, P. (2023). Caspase-4 Activation and Recruitment to Intracellular Gram-Negative Bacteria. In: Fink, S.L. (eds) Pyroptosis. Methods in Molecular Biology, vol 2641. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3040-2_5

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  • DOI: https://doi.org/10.1007/978-1-0716-3040-2_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3039-6

  • Online ISBN: 978-1-0716-3040-2

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