Advertisement

Detecting Release of Bacterial dsDNA into the Host Cytosol Using Fluorescence Microscopy

  • Roland Felix Dreier
  • José Carlos Santos
  • Petr BrozEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)

Abstract

Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types.

Keywords

cGAS dsDNA F. novicida Innate immunity Fluorescence confocal microscopy 

Notes

Acknowledgments

This work was supported by a Career Development Award (CDA00032/2015) from the Human Frontiers Science Program.

References

  1. 1.
    Broz P, Monack DM (2013) Newly described pattern recognition receptors team up against intracellular pathogens. Nat Rev Immunol 13:551–565CrossRefPubMedGoogle Scholar
  2. 2.
    Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140:805–820CrossRefPubMedGoogle Scholar
  3. 3.
    Paludan SR, Bowie AG (2013) Immune sensing of DNA. Immunity 38:870–880CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Deretic V, Saitoh T, Akira S (2013) Autophagy in infection, inflammation and immunity. Nat Rev Immunol 13:722–737CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Monroe KM, McWhirter SM, Vance RE (2010) Induction of type I interferons by bacteria. Cell Microbiol 12:881–890CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16(7):407–420CrossRefPubMedGoogle Scholar
  7. 7.
    Hornung V, Ablasser A, Charrel-Dennis M et al (2009) AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458:514–518CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Sun L, Wu J, Du F et al (2013) Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science (New York, NY) 339:786–791CrossRefGoogle Scholar
  9. 9.
    Unterholzner L, Keating SE, Baran M et al (2010) IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol 11:997–1004CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wu J, Sun L, Chen X et al (2013) Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339:826–830CrossRefPubMedGoogle Scholar
  11. 11.
    Tanaka Y, Chen ZJ (2012) STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci Signal 5:ra20CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Boxx GM, Cheng G (2016) The roles of type I interferon in bacterial infection. Cell Host Microbe 19:760–769CrossRefPubMedGoogle Scholar
  13. 13.
    Cai X, Chiu Y-H, Chen ZJ (2014) The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol Cell 54:289–296CrossRefPubMedGoogle Scholar
  14. 14.
    Oyston PCF, Sjostedt A, Titball RW (2004) Tularaemia: bioterrorism defence renews interest in Francisella tularensis. Nat Rev Microbiol 2:967–978CrossRefPubMedGoogle Scholar
  15. 15.
    Jones JW, Broz P, Monack DM (2011) Innate immune recognition of francisella tularensis: activation of type-I interferons and the inflammasome. Front Microbiol 2:16CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jones CL, Napier BA, Sampson TR et al (2012) Subversion of host recognition and defense systems by Francisella spp. Microbiol Mol Biol Rev 76:383–404CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Chong A, Wehrly TD, Nair V et al (2008) The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression. Infect Immun 76:5488–5499CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Geier H, Celli J (2011) Phagocytic receptors dictate phagosomal escape and intracellular proliferation of Francisella tularensis. Infect Immun 79:2204–2214CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Barker JR, Chong A, Wehrly TD et al (2009) The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence. Mol Microbiol 74:1459–1470CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Clemens DL, Ge P, Lee B-Y et al (2015) Atomic structure of T6SS reveals interlaced array essential to function. Cell 160:940–951CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Meunier E, Wallet P, Dreier RF et al (2015) Guanylate-binding proteins promote activation of the AIM2 inflammasome during infection with Francisella novicida. Nat Immunol 16:476–484CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Storek KM, Gertsvolf NA, Ohlson MB et al (2015) cGAS and Ifi204 cooperate to produce type I IFNs in response to Francisella infection. J Immunol 194:3236–3245CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Jones JW, Kayagaki N, Broz P et al (2010) Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. Proc Natl Acad Sci U S A 107:9771–9776CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Hansen K, Prabakaran T, Laustsen A et al (2014) Listeria monocytogenes induces IFNβ expression through an IFI16-, cGAS- and STING-dependent pathway. EMBO J 33:1654–1666CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Collins AC, Cai H, Li T et al (2015) Cyclic GMP-AMP synthase is an innate immune DNA sensor for mycobacterium tuberculosis. Cell Host Microbe 17:820–828CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Wassermann R, Gulen MF, Sala C et al (2015) Mycobacterium tuberculosis differentially activates cGAS- and inflammasome-dependent intracellular immune responses through ESX-1. Cell Host Microbe 17:799–810CrossRefPubMedGoogle Scholar
  27. 27.
    Watson RO, Bell SL, MacDuff DA et al (2015) The cytosolic sensor cGAS detects mycobacterium tuberculosis DNA to induce type I interferons and activate autophagy. Cell Host Microbe 17:811–819CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Stanley ER, Berg KL, Einstein DB et al (1997) Biology and action of colony–stimulating factor-1. Mol Reprod Dev 46:4–10CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Roland Felix Dreier
    • 1
  • José Carlos Santos
    • 1
  • Petr Broz
    • 1
    Email author
  1. 1.Focal Area Infection Biology, BiozentrumUniversity of BaselBaselSwitzerland

Personalised recommendations