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Intracellular bacteria engage a STING–TBK1–MVB12b pathway to enable paracrine cGAS–STING signalling

Nature Microbiology volume 4, pages 701–713 (2019)Cite this article

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Abstract

The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS)–stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.

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Fig. 1: Supernatants from cells infected with intracellular bacteria contain IFN-inducing potential.
Fig. 2: Foreign intracellular DNA stimulates IFN-β expression in bystander cells through EVs.
Fig. 3: Listeria infection activates EV-dependent stimulation of type I IFN expression in bystander cells.
Fig. 4: EVs from Listeria-infected cells augment apoptosis in T lymphocytes.
Fig. 5: The sorting of foreign DNA into EVs requires STING and TBK1.
Fig. 6: The sorting of foreign DNA into EVs requires TBK1-mediated phosphorylation of MVB12b.

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Data availability

The full next-generation sequencing dataset is available at the European Nucleotide Archive with the identifier ‘ena-STUDY-AARHUS UNIVERSITY 12-12-2018-17:12:31:528-124’, under accession number PRJEB30324. The full mass spectrometry dataset is available at https://www.ebi.ac.uk/pride/archive/projects/PXD012135. Original immunoblots are shown in Supplementary Fig. 7.

References

  1. Barbuddhe, S. B. & Chakraborty, T. Listeria as an enteroinvasive gastrointestinal pathogen. Curr. Top. Microbiol. Immunol. 337, 173–195 (2009).

    CAS  PubMed  Google Scholar 

  2. Cossart, P. et al. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect. Immun. 57, 3629–3636 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Rothe, J. et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364, 798–802 (1993).

    Article  CAS  Google Scholar 

  4. Edelson, B. T. & Unanue, E. R. MyD88-dependent but Toll-like receptor 2-independent innate immunity to Listeria: no role for either in macrophage listericidal activity. J. Immunol. 169, 3869–3875 (2002).

    Article  CAS  Google Scholar 

  5. Ladel, C. H., Flesch, I. E., Arnoldi, J. & Kaufmann, S. H. Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection. J. Immunol. 153, 3116–3122 (1994).

    CAS  PubMed  Google Scholar 

  6. Stockinger, S. et al. Production of type I IFN sensitizes macrophages to cell death induced by Listeria monocytogenes. J. Immunol. 169, 6522–6529 (2002).

    Article  CAS  Google Scholar 

  7. Auerbuch, V., Brockstedt, D. G., Meyer-Morse, N., O’Riordan, M. & Portnoy, D. A. Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes. J. Exp. Med. 200, 527–533 (2004).

    Article  CAS  Google Scholar 

  8. Carrero, J. A., Calderon, B. & Unanue, E. R. Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. J. Exp. Med. 200, 535–540 (2004).

    Article  CAS  Google Scholar 

  9. O’Connell, R. M. et al. Type I interferon production enhances susceptibility to Listeria monocytogenes infection. J. Exp. Med. 200, 437–445 (2004).

    Article  Google Scholar 

  10. Luecke, S. & Paludan, S. R. Molecular requirements for sensing of intracellular microbial nucleic acids by the innate immune system. Cytokine 98, 4–14 (2016).

    Article  Google Scholar 

  11. Kawai, T. & Akira, S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011).

    Article  CAS  Google Scholar 

  12. Kawai, T. et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol. 6, 981–988 (2005).

    Article  CAS  Google Scholar 

  13. Seth, R. B., Sun, L., Ea, C. K. & Chen, Z. J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-κB and IRF 3. Cell 122, 669–682 (2005).

    Article  CAS  Google Scholar 

  14. Ishikawa, H., Ma, Z. & Barber, G. N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009).

    Article  CAS  Google Scholar 

  15. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    Article  CAS  Google Scholar 

  16. Berg, R. K. et al. T cells detect intracellular DNA but fail to induce type I IFN responses: implications for restriction of HIV replication. PLoS ONE 9, e84513 (2014).

    Article  Google Scholar 

  17. Larkin, B. et al. Cutting edge: activation of STING in T cells induces type I IFN responses and cell death. J. Immunol. 199, 397–402 (2017).

    Article  CAS  Google Scholar 

  18. Cerboni, S. et al. Intrinsic antiproliferative activity of the innate sensor STING in T lymphocytes. J. Exp. Med. 214, 1769–1785 (2017).

    Article  CAS  Google Scholar 

  19. Gulen, M. F. et al. Signalling strength determines proapoptotic functions of STING. Nat. Commun. 8, 427 (2017).

    Article  Google Scholar 

  20. Hansen, K. et al. Listeria monocytogenes induces IFNβ expression through an IFI16-, cGAS- and STING-dependent pathway. EMBO J. 33, 1654–1666 (2014).

    Article  CAS  Google Scholar 

  21. Woodward, J. J., Iavarone, A. T. & Portnoy, D. A. c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science 328, 1703–1705 (2010).

    Article  CAS  Google Scholar 

  22. Abdullah, Z. et al. RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids. EMBO J. 31, 4153–4164 (2012).

    Article  CAS  Google Scholar 

  23. Czuczman, M. A. et al. Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread. Nature 509, 230–234 (2014).

    Article  CAS  Google Scholar 

  24. Schorey, J. S., Cheng, Y., Singh, P. P. & Smith, V. L. Exosomes and other extracellular vesicles in host–pathogen interactions. EMBO Rep. 16, 24–43 (2015).

    Article  CAS  Google Scholar 

  25. Trajkovic, K. et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319, 1244–1247 (2008).

    Article  CAS  Google Scholar 

  26. Kosaka, N. et al. Secretory mechanisms and intercellular transfer of microRNAs in living cells. J. Biol. Chem. 285, 17442–17452 (2010).

    Article  CAS  Google Scholar 

  27. Liu, S. et al. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science 347, aaa2630 (2015).

    Article  Google Scholar 

  28. Jeppesen, D. K. et al. Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J. Extracell. Vesicles 3, 25011 (2014).

    Article  Google Scholar 

  29. Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011).

    Article  CAS  Google Scholar 

  30. Ong, S. E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  31. Christ, L., Raiborg, C., Wenzel, E. M., Campsteijn, C. & Stenmark, H. Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem. Sci. 42, 42–56 (2017).

    Article  CAS  Google Scholar 

  32. Ablasser, A. et al. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 503, 530–534 (2013).

    Article  CAS  Google Scholar 

  33. Bridgeman, A. et al. Viruses transfer the antiviral second messenger cGAMP between cells. Science 349, 1228–1232 (2015).

    Article  CAS  Google Scholar 

  34. Gentili, M. et al. Transmission of innate immune signaling by packaging of cGAMP in viral particles. Science 349, 1232–1236 (2015).

    Article  CAS  Google Scholar 

  35. Robbins, P. D. & Morelli, A. E. Regulation of immune responses by extracellular vesicles. Nat. Rev. Immunol. 14, 195–208 (2014).

    Article  CAS  Google Scholar 

  36. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    Article  CAS  Google Scholar 

  37. Dreux, M. et al. Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity. Cell Host Microbe 12, 558–570 (2012).

    Article  CAS  Google Scholar 

  38. Yang, X. et al. Hepatitis B virus-encoded microRNA controls viral replication. J. Virol. 91, e01919–16 (2017).

    PubMed  PubMed Central  Google Scholar 

  39. Pegtel, D. M. et al. Functional delivery of viral miRNAs via exosomes. Proc. Natl Acad. Sci. USA 107, 6328–6333 (2010).

    Article  CAS  Google Scholar 

  40. Henry, T. et al. Type I IFN signaling constrains IL-17A/F secretion by γδ T cells during bacterial infections. J. Immunol. 184, 3755–3767 (2010).

    Article  CAS  Google Scholar 

  41. Gaidt, M. M. et al. The DNA inflammasome in human myeloid cells is initiated by a STING–cell death program upstream of NLRP3. Cell 171, 1110–1124 (2017).

    Article  CAS  Google Scholar 

  42. Paludan, S. R. & Bowie, A. G. Immune sensing of DNA. Immunity 38, 870–880 (2013).

    Article  CAS  Google Scholar 

  43. Fernandes-Alnemri, T. et al. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat. Immunol. 11, 385–393 (2010).

    Article  CAS  Google Scholar 

  44. Kim, S. et al. Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome. Eur. J. Immunol. 40, 1545–1551 (2010).

    Article  CAS  Google Scholar 

  45. Stockinger, S. et al. Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes. PLoS Pathog. 5, e1000355 (2009).

    Article  Google Scholar 

  46. Holm, C. K. et al. Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses. Nat. Commun. 7, 10680 (2016).

    Article  CAS  Google Scholar 

  47. Ogunjimi, B. et al. Inborn errors in RNA polymerase III underlie severe Varicella zoster virus infections. J. Clin. Invest. 127, 3543–3556 (2017).

    Article  Google Scholar 

  48. Skoble, J., Portnoy, D. A. & Welch, M. D. Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility. J. Cell Biol. 150, 527–538 (2000).

    Article  CAS  Google Scholar 

  49. Weiss, D. S. et al. In vivo negative selection screen identifies genes required for Francisella virulence. Proc. Natl Acad. Sci. USA 104, 6037–6042 (2007).

    Article  CAS  Google Scholar 

  50. Horan, K. A. et al. Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors. J. Immunol. 190, 2311–2319 (2013).

    Article  CAS  Google Scholar 

  51. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  52. Thery, C., Amigorena, S., Raposo, G. & Clayton, A. in Current Protocols in Cell Biology 3.22.1–3.22.29 (Wiley Online Library, 2006).

  53. Chevillet, J. R. et al. Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc. Natl Acad. Sci. USA 111, 14888–14893 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

The technical assistance of K. Stadel Petersen, the next-generation sequencing facility at the Department of Molecular Medicine, Aarhus University Hospital, and the FACS Core facility, Aarhus University is greatly appreciated. This work was funded by The Danish Medical Research Council (12-124330 to S.R.P.), Novo Nordisk Foundation (NNF18OC0030274 to S.R.P.), Lundbeck Foundation (R198-2015-171 to S.R.P.), European Research Council (786602 to S.R.P.), EU FP7 MOBILEX programme (DFF – 5053-00011 to R.N.), BMBF (JPI-AMR - FKZ 01Kl1702) and DFG (SFB/TR-84 TP C01) (both to B.S.), and Austrian Science Fund through grant P 25186-B22 (to T.D.).

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Authors and Affiliations

  1. Department of Biomedicine, Aarhus University, Aarhus, Denmark

    Ramya Nandakumar, Thaneas Prabakaran, Ensieh Farahani, Bao-cun Zhang, Sonia Assil, Christian K. Holm, Tanja Klause, Martin K. Thomsen & Søren R. Paludan

  2. Max F. Perutz Laboratories, Department of Microbiology, Immunobiology and Genetics, University of Vienna, Vienna, Austria

    Roland Tschismarov & Thomas Decker

  3. Experimental Systems Immunology, Max Planck Institute of Biochemistry, Munich, Germany

    Felix Meissner

  4. Department of Chemistry, Aarhus University, Aarhus, Denmark

    Abhichart Krissanaprasit & Kurt V. Gothelf

  5. Center for DNA Nanotechnology, Aarhus University, Aarhus, Denmark

    Abhichart Krissanaprasit & Kurt V. Gothelf

  6. Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark

    Abhichart Krissanaprasit, Kenneth A. Howard & Kurt V. Gothelf

  7. CIRI, Inserm, CNRS, Ecole Normale Supérieure Lyon and University Claude Bernard Lyon 1, Lyon, France

    Amandine Martin & Thomas Henry

  8. Institute for Lung Research, German Center for Lung Research, Universities of Giessen and Marburg Lung Centre, Philipps-University Marburg, Marburg, Germany

    Wilhelm Bertrams & Bernd Schmeck

  9. Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Andrea Ablasser

  10. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Kenneth A. Howard

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Contributions

R.N. and S.R.P. conceived the idea and designed the experiments. R.N., R.T., F.M., T.P., A.K., B.Z, S.A., A.M., W.B., A.A., T.K. and M.K.T. performed the experiments. F.M. designed, performed and analysed the phosphoproteomics experiment. E.F. analysed the next-generation sequencing data. C.K.H., B.S., K.A.H., T.H., K.V.G., T.D. and S.R.P. supervised the experiments. R.N. and S.R.P. wrote the manuscript.

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Correspondence to Søren R. Paludan.

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Supplementary Information

Supplementary Figures 1–7 and Supplementary Table 1.

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Supplementary Table 2

Full dataset for phosphoproteome analysis of dsDNA-stimulated cells.

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Nandakumar, R., Tschismarov, R., Meissner, F. et al. Intracellular bacteria engage a STING–TBK1–MVB12b pathway to enable paracrine cGAS–STING signalling. Nat Microbiol 4, 701–713 (2019). https://doi.org/10.1038/s41564-019-0367-z

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