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.
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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 OM982668–OM982674, respectively. Source data are available for all the main figures and for Extended Data Figs. 3, 4, 6 and 7.
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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).
R.S. is a scientific cofounder and advisor of BiomX and Ecophage. The remaining authors declare no competing interests.
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Extended data figures and tables
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.
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).
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.
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.
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).
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.
Map of the pSG-thrC-phSpank vector.
Map of the pJG_thrC_dCAS9_gRNA vector.
Map of the bdSumo_TAD1 plasmid.
Map of the pSG-thrC-Phspank-cmTad1 vector.
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Leavitt, A., Yirmiya, E., Amitai, G. et al. Viruses inhibit TIR gcADPR signalling to overcome bacterial defence. Nature 611, 326–331 (2022). https://doi.org/10.1038/s41586-022-05375-9