Skip to main content

Thank you for visiting 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.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence


The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1,2,3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are ‘sponges’ that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ′′–2′ glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1′′–3′ gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1′′–2′ gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Tad1 inhibits Thoeris defence.
Fig. 2: Tad1 proteins inhibit Thoeris defence.
Fig. 3: Tad1 cancels Thoeris-mediated defence by physically binding and sequestering the Thoeris-derived signalling molecule.
Fig. 4: Structure of Tad1 and identification of 1′′–2′ gcADPR.

Data availability

Data that support the findings of this study are available within the article and its Supplementary Tables and Supplementary files. IMG/MGV accessions, protein sequences and nucleotide sequences appear in Supplementary Tables 2–4. Coordinates and structure factors of cbTad1 apo and cbTad1–1''–2' gcADPR have been deposited in the PDB under accession codes 7UAV and 7UAW. The genome sequences of phages SBSphiJ1–SBSphiJ7 have been deposited with GenBank under accession codes OM982668OM982674, respectively. Source data are available for all the main figures and for Extended Data Figs. 3, 4, 6 and 7


  1. Ofir, G. et al. Antiviral activity of bacterial TIR domains via immune signalling molecules. Nature 600, 116–120 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Burch-Smith, T. M. & Dinesh-Kumar, S. P. The functions of plant TIR domains. Sci. STKE 2007, pe46 (2007).

    Article  PubMed  Google Scholar 

  3. Fitzgerald, K. A. & Kagan, J. C. Toll-like receptors and the control of immunity. Cell 180, 1044–1066 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wan, L. et al. TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death. Science 365, 799–803 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Horsefield, S. et al. NAD+ cleavage activity by animal and plant TIR domains in cell death pathways. Science 365, 793–799 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Ka, D., Oh, H., Park, E., Kim, J.-H. & Bae, E. Structural and functional evidence of bacterial antiphage protection by Thoeris defense system via NAD+ degradation. Nat. Commun. 11, 2816 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bayless, A. M. & Nishimura, M. T. Enzymatic functions for Toll/interleukin-1 receptor domain proteins in the plant immune system. Front. Genet. 11, 539 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Peters, J. M. et al. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165, 1493–1506 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, I. M. A. et al. IMG/M v.5.0: an integrated data management and comparative analysis system for microbial genomes and microbiomes. Nucleic Acids Res. 47, D666–D677 (2019).

    Article  CAS  PubMed  Google Scholar 

  11. Nayfach, S. et al. Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome. Nat. Microbiol. 6, 960–970 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Athukoralage, J. S. et al. An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity. Nature 577, 572–575 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hobbs, S. J. et al. Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity. Nature 605, 522–526 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Manik, M. K. et al. Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling. Science 377, eadc8969 (2022).

  15. Weagley, J. S. et al. Products of gut microbial Toll/interleukin-1 receptor domain NADase activities in gnotobiotic mice and Bangladeshi children with malnutrition. Cell Rep. 39, 110738 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yu, D. et al. TIR domains of plant immune receptors are 2′,3′-cAMP/cGMP synthetases mediating cell death. Cell 185, 2370–2386 (2022).

    Article  CAS  PubMed  Google Scholar 

  17. Huang, S. et al. Identification and receptor mechanism of TIR-catalyzed small molecules in plant immunity. Science 377, eabq3297 (2022).

    Article  CAS  PubMed  Google Scholar 

  18. Jia, A. et al. TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity. Science 377, eabq8180 (2022).

    Article  CAS  PubMed  Google Scholar 

  19. Tal, N. et al. Cyclic CMP and cyclic UMP mediate bacterial immunity against phages. Cell 184, 5728–5739 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Whiteley, A. T. et al. Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cohen, D. et al. Cyclic GMP-AMP signalling protects bacteria against viral infection. Nature 574, 691–695 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Essuman, K., Milbrandt, J., Dangl, J. L. & Nishimura, M. T. Shared TIR enzymatic functions regulate cell death and immunity across the tree of life. Science 377, eabo0001 (2022).

    Article  CAS  PubMed  Google Scholar 

  23. Johnson, A. G. et al. Bacterial gasdermins reveal an ancient mechanism of cell death. Science 375, 221–225 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bernheim, A. et al. Prokaryotic viperins produce diverse antiviral molecules. Nature 589, 120–124 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Morehouse, B. R. et al. STING cyclic dinucleotide sensing originated in bacteria. Nature 586, 429–433 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kropinski, A. M., Mazzocco, A., Waddell, T. E., Lingohr, E. & Johnson, R. P. In Bacteriophages: Methods and Protocols (eds Clokie, M. R. J. & Kropinski, A. M.) 69–76 (Humana, 2009).

  27. Baym, M. et al. Inexpensive multiplexed library preparation for megabase-sized genomes. PLoS ONE 10, e0128036 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10 (2011).

    Article  Google Scholar 

  29. Nurk, S. et al. Assembling genomes and mini-metagenomes from highly chimeric reads. Res. Comput. Mol. Biol. 7821, 158–170 (2013).

  30. Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Mazzocco, A., Waddell, T. E. & Lingohr, R. P. J. In Bacteriophages: Methods and Protocols (eds Clokie, M. R. J. & Kropinski, A. M.) 81–85 (Humana, 2009).

  32. Steinegger, M. & Söding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026–1028 (2017).

    Article  CAS  PubMed  Google Scholar 

  33. Deatherage, D. E. & Barrick, J. E. In Engineering and Analyzing Multicellular Systems (eds Sun, L. & Shou, W.) 165–188 (Humana, 2014).

  34. Garb, J. et al. Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion. Nat. Microbiol. (2022).

  35. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Unger, T., Jacobovitch, Y., Dantes, A., Bernheim, R. & Peleg, Y. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J. Struct. Biol. 172, 34–44 (2010).

    Article  CAS  PubMed  Google Scholar 

  39. Frey, S. & Görlich, D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. J. Chromatogr. A 1337, 95–105 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Peleg, Y. & Unger, T. In Structural Proteomics (eds Kobe, B., Guss, M. & Huber, T.) 197–208 (Humana, 2008).

  41. Zhou, W. et al. Structure of the human cGAS-DNA complex reveals enhanced control of immune surveillance. Cell 174, 300–311 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Eaglesham, J. B., Pan, Y., Kupper, T. S. & Kranzusch, P. J. Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling. Nature 566, 259–263 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  45. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  47. Weiss, M. S. Global indicators of X-ray data quality. J. Appl. Crystallogr. 34, 130–135 (2001).

    Article  CAS  Google Scholar 

  48. Gilchrist, C. L. M. & Chooi, Y.-H. clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics 37, 2473–2475 (2021).

    Article  CAS  Google Scholar 

Download references


We thank the Sorek laboratory members for comments on the manuscript and fruitful discussion. We also thank Y. Peleg and S. Albeck from the Center for Structural Proteomics within the Weizmann Institute of Science for assistance with protein expression, M. Danielsen and D. Malheiro from MS-Omics for conducting MS experiments, S. C. Wilson for advice on gcADPR analysis and K. Arnett from Harvard University’s Center for Macromolecular Interactions for assistance with the ITC experiments. R.S. was supported, in part, by the European Research Council (grant no. ERC-AdG GA 101018520), Israel Science Foundation (grant no. ISF 296/21), Deutsche Forschungsgemeinschaft (SPP 2330, grant no. 464312965), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, the Minerva Foundation with funding from the Federal German Ministry for Education and Research and the Knell Family Center for Microbiology. P.J.K. was supported, in part, by the Pew Biomedical Scholars programme and The Mathers Foundation. E.Y. is supported by the Israeli Council for Higher Education (CHE) via the Weizmann Data Science Research Center, B.R.M. is supported as a Ruth L. Kirschstein NRSA Postdoctoral Fellow (no. NIH F32GM133063) and S.J.H. is supported through a Cancer Research Institute Irvington Postdoctoral Fellowship (no. CRI3996). X-ray data were collected at Northeastern Collaborative Access Team beamlines 24-ID-C and 24-ID-E (no. P30 GM124165), including use of a Pilatus detector (no. S10RR029205), an Eiger detector (no. S10OD021527) and the Argonne National Laboratory Advanced Photon Source (no. DE-AC02-06CH11357).

Author information

Authors and Affiliations



A. Leavitt isolated phages and conducted all in vivo experiments. E.Y. built and executed the computational pipeline and analysed data. G.A. performed biochemical experiments with cell lysates and led the mechanistic characterization of Tad1 activity. A. Lu performed the structural analysis of Tad1 and biochemically and structurally characterized TIR-derived signalling molecules. J.G. designed and helped with performing knock-in and knockdown experiments. E.H. helped with LC–MC data analysis. B.R.M. and S.J.H. helped with structural analysis and biochemical characterization of TIR-derived molecules. S.P.A. helped with ITC data collection and stoichiometry calculations. Z.-Y.J.S. performed NMR analysis. The study was supervised by P.J.K. and R.S. The manuscript was written by E.Y., A. Lu, P.J.K. and R.S. All authors contributed to editing the manuscript and support the conclusions.

Corresponding authors

Correspondence to Philip J. Kranzusch or Rotem Sorek.

Ethics declarations

Competing interests

R.S. is a scientific cofounder and advisor of BiomX and Ecophage. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature thanks Joseph Bondy-Denomy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Genome comparison of eight phages from the SBSphiJ group.

Amino acid sequence similarity between the ORFs is marked by grey shading. Genome similarity was visualized using clinker48.

Extended Data Fig. 2 Multiple sequence alignment of the original Tad1 from phage SBSphiJ7, and 10 Tad1 homologs that were verified experimentally as anti Thoeris proteins.

The strength of shading indicates degree of residue conservation. The determined Clostridium botulinum ATCC 9564 (orange highlight) Tad1 secondary structure is depicted below, conserved loops involved in ligand-binding are boxed in black, and ligand-binding residues are marked with arrows.

Extended Data Fig. 3 Lysates derived from cells infected by phage SBSphiJ7 do not activate ThsA.

Cells expressing ThsB (native promoter) were infected with phage SBSphiJ or phage SBSphiJ7 at multiplicity of infection (MOI) of 5. Control cells that do not express ThsB were infected with phage SBSphiJ. Shown is the activation of ThsA NADase activity by lysates from the infected cells. Purified ThsA used in this experiment is from a different batch than that used in Fig. 3, and hence the background activity of ThsA is different in the two assays. Bars represent the mean of three experiments, with individual data points overlaid. Asterisk marks a statistically significant difference in NADase activity (one-way ANOVA, P = 0.039).

Source data

Extended Data Fig. 4 Purification and characterization of 1′′–2′ gcADPR from BdTIR.

a, HPLC analysis of filtered lysate from E. coli expressing a strep-tagged BdTIR after treatment with buffer, cmTad1, or cmTad1 and subsequent boiling. cmTad1 selectively binds, and upon boiling, releases 1′′–2′ gcADPR. b, Filtered lysates from panel a were diluted 1:20 for stimulation of ThsA NADase activity. Data from three replicates are presented. c, Superdex 75 16/600 size-exclusion chromatography of 1′′–2′ gcADPR-bound or apo state cmTad1 purified from cells expressing BdTIR or only cmTad1, respectively. 1′′–2′ gcADPR-bound cmTad1 shows an ~0.8 mL right shift compared to cmTad1 in the apo state. d, A260/A280 signal ratios of apo state and 1′′–2′ gcADPR-bound cmTad1 in folding (25 °C) or denaturing (95 °C) conditions. 1′′–2′ gcADPR-bound cmTad1 shows a higher A260/A280 ratio and yields high A260 absorbance upon heat denaturation. Data are presented as mean values +/− SEM. e, HPLC analysis demonstrating that BdTIR-derived, cmTad1-purified 1′′–2′ gcADPR migrates as a single, unique peak. 1′′–2′ gcADPR separates from relevant molecule standards at pH = 5.1. f, Isothermal titration calorimetry measurement of cmTad1 affinity for 1′′–2′ gcADPR. Data shown are representative of three individual experiments.

Source data

Extended Data Fig. 5 NMR analysis of 1′′–2′ gcADPR from BdTIR.

a, 13C-HSQC spectrum of 1′′–2′ gcADPR showing assignments of the two ribose rings. Extraneous peaks from glycerol contaminants are marked with * symbols. b, 13C-HMBC spectrum (red) superimposed on to the 13C-HSQC spectrum (blue/green). The 3-bond J-coupled cross peaks between the 2′ and 1′′ positions of the two ribose rings are shown with black arrows. c, 13C-HMBC spectrum (red) showing the 3-bond J-coupled peak (marked with a blue arrow) between 13C resonance of carbon 8 and the 1H resonance of the 1' proton. d, 15N-HMBC spectrum showing the 3-bond J-coupled peak between 15N resonance of nitrogen 9 and 1H resonance of 2′ proton, aligned with the 2-bond J-coupled peak between 15N resonance of nitrogen 9 and 1H resonance of proton 8.

Extended Data Fig. 6 Purification of the cADPR isomer from ThsB′ TIR domain and comparison to 1′′–2′ gcADPR.

a, Purified ThsB′ (methods) was incubated with NAD+ and the reaction products were filtered and analyzed by HPLC. b, Y-axis zoom of (a). The predicted ThsA-activating ThsB′ reaction product (ThsB′ cADPR isomer) is indicated with an arrow. c, HPLC analysis of the ThsB′ cADPR isomer reaction after addition of cmTad1 followed by concentration and heat denaturation demonstrates that cmTad1 is able to purify and enrich the predicted ThsB′ cADPR isomer peak. This peak was further isolated by HPLC fractionation. d, HPLC analysis shows that the cmTad1-purified ThsB′ cADPR isomer migrates as a unique peak with a small amount of residual NAD+. ThsB′ cADPR isomer is distinct from 1′′–2′ gcADPR and separates from relevant molecule standards at pH = 6.8. (e-f) ThsA NADase activation curves of 1′′–2′ gcADPR (e) and 1′′–3′ gcADPR (f). ThsA activation is ~50–100× more sensitive to 1′′–3′ gcADPR than 1′′–2′ gcADPR.

Source data

Extended Data Fig. 7 The Thoeris ThsB-derived cADPR isomer compared to 1′′–3′ gcADPR.

a, Purified AaTIRTIR was incubated with NAD+, and the reaction products were filtered, treated with buffer, cmTad1, or cmTad1 with subsequent boiling, and analyzed by HPLC. b, Y-axis zoom of panel (a). cmTad1 selectively binds, and upon boiling, releases the AaTIRTIR product, 1′′–3′ gcADPR. c, Filtered reaction products from panel (a) were diluted 1:1000 and tested for activation of ThsA NADase activity. d, HPLC analysis of purified ThsB′ cADPR isomer and the fractionated AaTIRTIR reaction shows consistent retention times for 50 μM 1′′–3′ gcADPR, 50 μM ThsB′ cADPR isomer, and a 50 μM equimolar mixture of the two, demonstrating that ThsB′ produces 1′′–3′ gcADPR. e, Overlay of traces from (d).

Source data

Extended Data Table 1 Summary of crystallography data collection, phasing and refinement statistics
Extended Data Table 2 Atomic assignment of NMR peaks

Supplementary information

Reporting Summary

Peer Review File

Supplementary Tables

This file contains Supplementary Tables 1–5. Supplementary Table 1: sequence similarity between alignable regions of SBSphiJ-like phages. Supplementary Table 2: candidate genes from SBSphiJ7 that were cloned and tested. Supplementary Table 3: Tad1 homologues from the IMG database. Supplementary Table 4: Tad1 homologues from the MGV database. Supplementary Table 5: primers used for cmTad1 ligand-binding site residue substitution.

Supplementary Data 1

Map of the pSG-thrC-phSpank vector.

Supplementary Data 2

Map of the pJG_thrC_dCAS9_gRNA vector.

Supplementary Data 3

Map of the bdSumo_TAD1 plasmid.

Supplementary Data 4

Map of the pSG-thrC-Phspank-cmTad1 vector.

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Leavitt, A., Yirmiya, E., Amitai, G. et al. Viruses inhibit TIR gcADPR signalling to overcome bacterial defence. Nature 611, 326–331 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


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.


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