Advertisement

Methods for Detection of Pyrin Inflammasome Assembly in Macrophages Infected with Yersinia spp.

  • Natasha P. Medici
  • James B. BliskaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2010)

Abstract

The Yersinia effector proteins YopE and YopT are important bacterial virulence factors that are secreted into infected host cells and can inactivate Rho GTPases, like RhoA, Rac1, and Cdc42. In order to compensate for the consequences of this effect, the host cell can sense RhoA modifications and trigger a proinflammatory reaction to control the infection. This host response, known as pyrin inflammasome assembly, is normally prevented by another important effector, YopM, allowing Yersinia to counteract this conserved innate immune response. Once assembled, the pyrin inflammasome can activate caspase-1 via proteolysis, leading to IL-1β secretion and cell death through pyroptosis. Here we describe how to measure pyrin inflammasome assembly, in response to YopE or YopT activities, when macrophages are infected with yopM mutant Yersinia. Using primary mouse macrophages as host cells, we show how to detect this host response through the downstream events of pyrin dephosphorylation, caspase-1 proteolysis, IL-1β release, and pyroptosis.

Key words

Yersinia Effectors Toxins Inflammasome Pyrin Inflammation Rho GTPases 

Notes

Acknowledgments

The authors thank Nicole Loeven and Amrapali Ghosh for editing the manuscript. The preparation of this publication was supported by the NIH under award R01AI099222 (JB) and by Science Without Borders/CAPES—Brazil (NM).

References

  1. 1.
    Cornelis GR, Wolf‐Watz H (1997) The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol Microbiol 23(5):861–867.  https://doi.org/10.1046/j.1365-2958.1997.2731623.xCrossRefPubMedGoogle Scholar
  2. 2.
    Cornelis GR (2002) Yersinia type III secretion: send in the effectors. J Cell Biol 158(3):401–408.  https://doi.org/10.1083/jcb.200205077CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Viboud GI, Bliska JB (2005) Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis. Annu Rev Microbiol 59:69–89.  https://doi.org/10.1146/annurev.micro.59.030804.121320CrossRefPubMedGoogle Scholar
  4. 4.
    Black DS, Bliska JB (2000) The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence. Mol Microbiol 37(3):515–527CrossRefGoogle Scholar
  5. 5.
    Shao F, Vacratsis PO, Bao Z, Bowers KE, Fierke CA, Dixon JE (2003) Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases. Proc Natl Acad Sci U S A 100(3):904–909.  https://doi.org/10.1073/pnas.252770599CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Xu H, Yang J, Gao W, Li L, Li P, Zhang L, Gong Y-N, Peng X, Xi JJ, Chen S, Wang F, Shao F (2014) Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 513(7517):237–241.  https://doi.org/10.1038/nature13449CrossRefPubMedGoogle Scholar
  7. 7.
    Chung LK, Park YH, Zheng Y, Brodsky IE, Hearing P, Kastner DL, Chae JJ, Bliska JB (2016) The Yersinia virulence factor YopM hijacks host kinases to inhibit type III effector-triggered activation of the pyrin inflammasome. Cell Host Microbe 20(3):296–306.  https://doi.org/10.1016/j.chom.2016.07.018CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ratner D, Orning MPA, Proulx MK, Wang D, Gavrilin MA, Wewers MD, Alnemri ES, Johnson PF, Lee B, Mecsas J, Kayagaki N, Goguen JD, Lien E (2016) The Yersinia pestis effector YopM inhibits pyrin inflammasome activation. PLoS Pathog 12(12):e1006035.  https://doi.org/10.1371/journal.ppat.1006035CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Chae JJ, Wood G, Masters SL, Richard K, Park G, Smith BJ, Kastner DL (2006) The B30.2 domain of pyrin, the familial Mediterranean fever protein, interacts directly with caspase-1 to modulate IL-1beta production. Proc Natl Acad Sci U S A 103(26):9982–9987.  https://doi.org/10.1073/pnas.0602081103CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Gao W, Yang J, Liu W, Wang Y, Shao F (2016) Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation. Proc Natl Acad Sci U S A 113(33):E4857–E4866.  https://doi.org/10.1073/pnas.1601700113CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Masters SL, Lagou V, Jéru I, Baker PJ, Van Eyck L, Parry DA, Lawless D, De Nardo D, Garcia-Perez JE, Dagley LF, Holley CL, Dooley J, Moghaddas F, Pasciuto E, Jeandel P-Y, Sciot R, Lyras D, Webb AI, Nicholson SE, De Somer L, van Nieuwenhove E, Ruuth-Praz J, Copin B, Cochet E, Medlej-Hashim M, Megarbane A, Schroder K, Savic S, Goris A, Amselem S, Wouters C, Liston A (2016) Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation. Sci Transl Med 8(332):332ra345CrossRefGoogle Scholar
  12. 12.
    Park YH, Wood G, Kastner DL, Chae JJ (2016) Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS. Nat Immunol 17(8):914–921.  https://doi.org/10.1038/ni.3457. http://www.nature.com/ni/journal/v17/n8/abs/ni.3457.html#supplementary-informationCrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Shin S, Brodsky IE (2015) The inflammasome: learning from bacterial evasion strategies. Semin Immunol 27(2):102–110.  https://doi.org/10.1016/j.smim.2015.03.006CrossRefPubMedGoogle Scholar
  14. 14.
    Shi J, Gao W, Shao F (2017) Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem Sci 42(4):245–254.  https://doi.org/10.1016/j.tibs.2016.10.004CrossRefPubMedGoogle Scholar
  15. 15.
    Kovacs SB, Miao EA (2017) Gasdermins: effectors of pyroptosis. Trends Cell Biol 27(9):673–684.  https://doi.org/10.1016/j.tcb.2017.05.005CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jorgensen I, Miao EA (2015) Pyroptotic cell death defends against intracellular pathogens. Immunol Rev 265(1):130–142.  https://doi.org/10.1111/imr.12287CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    von Moltke J, Ayres JS, Kofoed EM, Chavarría-Smith J, Vance RE (2013) Recognition of bacteria by inflammasomes. Annu Rev Immunol 31(1):73–106.  https://doi.org/10.1146/annurev-immunol-032712-095944CrossRefGoogle Scholar
  18. 18.
    Aubert Daniel F, Xu H, Yang J, Shi X, Gao W, Li L, Bisaro F, Chen S, Valvano Miguel A, Shao F (2016) A burkholderia type VI effector deamidates rho GTPases to activate the pyrin inflammasome and trigger inflammation. Cell Host Microbe 19(5):664–674.  https://doi.org/10.1016/j.chom.2016.04.004CrossRefPubMedGoogle Scholar
  19. 19.
    Schoberle TJ, Chung LK, McPhee JB, Bogin B, Bliska JB (2016) Uncovering an important role for YopJ in the inhibition of caspase-1 in activated macrophages and promoting Yersinia pseudotuberculosis virulence. Infect Immun 84(4):1062–1072.  https://doi.org/10.1128/iai.00843-15CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular Genetics and Microbiology, Center for Infectious DiseasesStony Brook UniversityStony BrookUSA
  2. 2.Department of Microbiology and ImmunologyGeisel School of Medicine at DartmouthHanoverUSA

Personalised recommendations