Abstract
Prokaryotic anti-phage immune systems use TIR and cGAS-like enzymes to produce 1′′-3′-glycocyclic ADP-ribose (1′′-3′-gcADPR) and cyclic dinucleotide (CDN) and cyclic trinucleotide (CTN) signalling molecules, respectively, which limit phage replication1,2,3. However, how phages neutralize these distinct and common systems is largely unclear. Here we show that the Thoeris anti-defence proteins Tad14 and Tad25 both achieve anti-cyclic-oligonucleotide-based anti-phage signalling system (anti-CBASS) activity by simultaneously sequestering CBASS cyclic oligonucleotides. Apart from binding to the Thoeris signals 1′′-3′-gcADPR and 1′′-2′-gcADPR, Tad1 also binds to numerous CBASS CDNs and CTNs with high affinity, inhibiting CBASS systems that use these molecules in vivo and in vitro. The hexameric Tad1 has six binding sites for CDNs or gcADPR, which are independent of the two high-affinity binding sites for CTNs. Tad2 forms a tetramer that also sequesters various CDNs in addition to gcADPR molecules, using distinct binding sites to simultaneously bind to these signals. Thus, Tad1 and Tad2 are both two-pronged inhibitors that, alongside anti-CBASS protein 2 (Acb26,7,8), establish a paradigm of phage proteins that use distinct binding sites to flexibly sequester a considerable breadth of cyclic nucleotides.
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Data availability
The coordinate and structure factors reported in this paper have been deposited at the PDB: 8KBB (apo CmTad1), 8KBC (CmTad1–cA3), 8KBD (CmTad1–cAAG), 8KBE (CbTad1–1′′,3′-gcADPR), 8KBF (CbTad1–1′′,3′-gcADPR–cA3), 8KBG (CbTad1–2′,3′-cGAMP), 8KBH (CbTad1–2′,3′-cGAMP–cA3), 8KBI (apo HgmTad2), 8KBJ (HgmTad2-1′′,2′-gcADPR) 8KBK (HgmTad2–1′′,2′-gcADPR-cGG), 8KBL (HgmTad2–1′′-3′-gcADPR–cGG), 8KBM (HgmTad2–cGG), 8WJC (HgmTad2–3′,3′-cGAMP), 8WJD (SptTad2–cGG) and 8WJE (apo SPO1 Tad2). All other data are available in the Article, Extended Data Figs. 1–11, Extended Data Table 1 and the Supplementary Information. Source data are provided with this paper.
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This paper does not report original code.
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Acknowledgements
We thank the staff at beamlines BL02U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility for their assistance with data collection; the staff at the Tsinghua University Branch of China National Center for Protein Sciences Beijing and S. Fan for providing facility support for X-ray diffraction of the crystal samples; and Y. Chen, Z. Yang and B. Zhou at the Institute of Biophysics, Chinese Academy of Sciences for technical help with ITC and SPR experiments. Y.F. is supported by National key research and development program of China (2022YFC3401500 and 2022YFC2104800), the National Natural Science Foundation of China (32371329 and 32171274), Beijing Nova Program (20220484160) and the Fundamental Research Funds for the Central Universities (QNTD2023-01). E.H. is supported by the National Science Foundation Graduate Research Fellowship Program (grant no. 2038436). J.B.-D.is supported by the US National Institutes of Health (R21AI168811 supported CBASS/Thoeris experiments; R01GM127489 supported CRISPR-Cas9 experiments), the Vallee Foundation and the Searle Scholarship. Any opinions, findings and conclusions or recommendations expressed in this Article are those of the authors and do not necessarily reflect the views of the National Science Foundation.
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Y.F. and J.B.-D. conceived and supervised the project and designed experiments. D.L., W.X., Y.W., X.L., Z.G., X.Z. and X.C. purified the proteins, grew and optimized the crystals, collected the diffraction data, and performed in vitro activity analysis and binding assays. Y.X. solved the crystal structures with the help of Y.F. and Y.Z.; I.F. performed in vivo phage experiments, strain engineering and Tad1/Tad2 protein bioinformatics. J.R. performed HPLC assays. E.H. performed cyclase bioinformatics. Y.F. wrote the original manuscript. J.B.-D., Y.F., I.F. and E.H. revised the manuscript.
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J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics, a consultant to LeapFrog Bio and BiomX, and a scientific advisory board member and co-founder of Acrigen Biosciences and ePhective Therapeutics. The J.B.-D. laboratory received prior research support from Felix Biotechnology.
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Extended data figures and tables
Extended Data Fig. 1 Bacterial hosts that Tad-encoding phage likely infect contain multiple CBASS CD-NTases that produce cyclic oligonucleotides.
Bacteria from the genus (a) Clostridium, (b) Bacteroides, (c) Sphingobacterium, and (d) Bacillus cereus group contain CBASS CD-NTases that produce cyclic oligonucleotides (c-oligos) with strong, weak, or no binding affinity to the Tad proteins tested in this study. The relative frequency of CD-NTases is quantified as the number of CD-NTase from a specific clade divided by the total number of CD-NTases identified using the NCBI blastp (see Methods for details). The legend indicates the c-oligos that are known or predicted to be produced by the indicated CBASS CD-NTases (Whiteley et al.22; Ye et al.34; Morehouse et al.40; Fatma et al.23). CD-NTases with currently unknown nucleotide products are indicated in the legend and the graphs.
Extended Data Fig. 2 Structural comparison between CbTad1 and CmTad1.
a, The hexameric form of CbTad1 are shown in cartoon model. Left: CbTad1 (PDB code: 7UAV). Right: CbTad1-1″−2′ gcADPR (PDB code: 7UAW). b, Structural comparison between CbTad1 and CmTad1 hexamers. Left: structural comparison between apo CbTad1 (coloured as in a) and apo CmTad1 (coloured orange). Right: structural comparison between CbTad1-1″−2′ gcADPR (coloured as in a) and apo CmTad1 (coloured orange). c, Detailed binding in the hexamer interface of CbTad1. Residues involved in hexamer formation are shown as sticks. Red dashed lines represent polar interactions. d, Sequence alignment between Tad1 homologues. The cyclic trinucleotide (CTN) and cyclic dinucleotides (CDN)/gcADPR binding sites are marked in yellow and red, respectively. Representative sequences were intentionally selected to show CDN/CTN binding site mutations. Residues involved in hexamer formation are marked with blue triangles. e, SLS studies of purified CbTad1 and its E100A/E101A/M104A/W105A/R108A mutant. Calculated molecular weight is shown above the peaks. Profile of the molecular standard sample is coloured in dark blue. f, Structural comparison between the dimer form of CbTad1 and CmTad1. Apo CbTad1 (PDB code: 7UAV) is coloured green. Apo CmTad1 is coloured in pink and magenta for the two protomers.
Extended Data Fig. 3 Tad1 binds to 2′3′-/3′,2′-cGAMP using the same binding pocket as gcADPR molecules.
a, Overall structure of CbTad1 hexamer bound to 2′,3′-cGAMP, which is shown as yellow sticks. b, Structural superimposition of apo, 1″−3′ gcADPR-bound and 2′,3′-cGAMP-bound CbTad1 protein. 1″−3′ gcADPR and 2′,3′-cGAMP are shown as orange and yellow sticks, respectively. The two loops that undergo conformational changes upon ligand binding are highlighted. c, Detailed binding between CbTad1 and 2′,3′-cGAMP. Residues involved in 2′,3′-cGAMP binding are shown as sticks. Red dashed lines represent polar interactions. 2Fo-Fc electron density of 2′,3′-cGAMP within one binding pocket is shown and contoured at 1 σ. d, ITC assays to test binding of 2′,3′ –cGAMP to CbTad1 mutants. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n = 3 independent experiments). Raw data for these curves are shown in Supplementary Fig. 3. e, Native PAGE showed the binding of CbTad1 and its mutants to 2′,3′-cGAMP. For gel source data, see Supplementary Fig. 1. f, Overall structure of CmTad1 hexamer complexed with cA3 and 2′,3′-cGAMP. cA3 and 2′,3′-cGAMP are shown as green and yellow sticks, respectively. Two views are shown. 2Fo-Fc electron density of cA3 and 2′,3′-cGAMP within CbTad1 hexamer contoured at 1 σ.
Extended Data Fig. 4 Binding of 1″−3′-gcADPR by CbTad1.
a, Overall structure of CbTad1 hexamer bound to 1″−3′ gcADPR, which is shown as orange sticks. b, Detailed binding between CbTad1 and 1″−3′ gcADPR. Residues involved in 1″−3′ gcADPR binding are shown as sticks. Red dashed lines represent polar interactions. 2Fo-Fc electron density of 1″−3′ gcADPR within one binding pocket is shown and contoured at 1 σ. c, Native PAGE showed the binding of CbTad1 mutants to cA3 and 1″−2′ gcADPR. For gel source date, see Supplementary Fig. 1. d, ITC assays to test binding of cA3 to CbTad1 mutant. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n = 3 independent experiments). Raw data for these curves are shown in Supplementary Fig. 3.
Extended Data Fig. 5 Mutations in independent nucleotide binding sites of SBS Tad1 disrupt specific inhibitory activities.
a, Plaque assay to test the activity of SBS Tad1 against Thoeris immunity in vivo. F10 phage was spotted in 10-fold serial dilutions on a lawn of P. aeruginosa cells expressing Thoeris operon genes (PAO1:Tn7 Thoeris SIR2), or without Thoeris (PAO1:Tn7 empty), electroporated with pHERD30T plasmids carrying SBS tad1 (wild type or mutant gene) or empty vector. b, Plaque assay to test the activity of SBS Tad1 against CBASS II-AGA immunity in vivo. PaMx41∆acb2 was spotted on a lawn of Pa011 cells with deletion of CBASS operon (Pa011ΔCBASS II-AGA) or Pa011 wild type cells (Pa011 wt), electroporated with pHERD30T plasmids carrying SBS Tad1 (wild type or mutant gene) or empty vector. c, Plaque assay to test the activity of SBS Tad1 against CBASS III-CAAA immunity in vivo. JBD67Δacb2 phage was spotted in 10-fold serial dilutions on a lawn of P. aeruginosa cells expressing Pa278 CBASS operon genes (PAO1:Tn7CBASS III-CAAA), or without the system (PAO1:Tn7 empty), electroporated with pHERD30T plasmids carrying SBS tad1 (wild type or mutant gene) or empty vector.
Extended Data Fig. 6 PhiKZ gp184 is Tad1 sponge protein that binds CDN/gcADPR molecules.
a, Sequence alignment among phiKZ Tad1 homologues and SBS Tad1. For Tad1 proteins from phiKZ, PA7, phiPA3, Phabio phages the sequences without N-terminal domain (putative packaging domains) were used for alignment. CDN/gcADPR binding sites are shown with red frames, CTN binding sites are shown with yellow frames. Arrows indicate mutations in the binding sites. The enumeration of start and end amino acid positions is shown for phiKZ gp184. b, ColabFold prediction of phiKZ gp184 structure. The Tad1 domain is in gold, and the putative packaging domain is in blue. The protease cleavage site position H109 is indicated with a red arrow. c, ITC assays to test binding of 3′,3′-cGAMP, 1″,3′- and 1″,2′-gcADPR to phiKZ Tad1. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n = 3 independent experiments). Raw data for these curves are shown in Supplementary Fig. 9. d, Native PAGE showed the binding of phiKZ Tad1 to cyclic nucleotides and gcADPR molecules. For gel source date, see Supplementary Fig. 1. e, Plaque assay to test the activity of phiKZ gp184 against CBASS II-AGA immunity in vivo. phiKZ∆gp184 was spotted on a lawn of P. aeruginosa cells expressing Pa278 CBASS operon genes (PAO1:Tn7CBASS III-CAAA), Pa011 CBASS operon genes (PAO1:Tn7CBASS II-AGA), Thoeris SIR2 operon genes (PAO1:Tn7 Thoeris I) or without the system (PAO1:Tn7 empty). Cells were electroporated with pHERD30T plasmids carrying phiKZ gp184 (109–230 amino acids) or empty vector.
Extended Data Fig. 7 SPO1 Tad2 does not bind to cyclic dinucleotides.
a, Native PAGE showed the binding of SPO1 Tad2 to cyclic nucleotides and gcADPR molecules. For gel source date, see Supplementary Fig. 1. b, The ability of SPO1 Tad2 to bind and release 3′,3′-cGAMP when treated with proteinase K was analysed by HPLC. 3′,3′-cGAMP was used as a control. The remaining nucleotides after incubation with SPO1 Tad2 was tested. c, Overlay of sensorgrams from surface plasmon resonance (SPR) experiments, used to determine kinetics of SPO1 Tad2 binding to cyclic dinucleotides. Data were fit with a model describing one-site binding for the ligands (black lines).
Extended Data Fig. 8 HgmTad2 binds to cGG and gcADPR molecules.
a, Profile of ion exchange chromatography of HgmTad2 using Resource Q column (1 mL, GE Healthcare). Proteins in peaks 1–3 are collected separately and marked as State 1–3. The proteins in three states were then subjected to native PAGE and SDS-PAGE, respectively. b, Native PAGE showed the binding of HgmTad2 in three states to 1″,2′ gcADPR. For gel source date of a and b, see Supplementary Fig. 1. c, SPR analysis of HgmTad2 binding to 2′,3′-cGAMP. The data was fitted with affinity model and the calculated KD was shown.
Extended Data Fig. 9 The binding pockets of 1″−2′ gcADPR and cGG.
a, The binding pocket of 1″−2′ gcADPR in the HgmTad2-1″−2′ gcADPR structure. 2Fo-Fc electron density of 1″−2′ gcADPR is shown and contoured at 1 σ. b, Structural superposition among HgmTad2 in the apo form (two types of conformations) and 1″−2′ gcADPR-bound form. HgmTad2 in the apo form is coloured orange and green for two types of conformations, respectively. HgmTad2 in the 1″−2′ gcADPR-bound form is coloured cyan and pink for the two protomers. c, Native PAGE showed the binding of HgmTad2 and its mutants to 1″−2′ gcADPR. d, The binding pocket of 3′,3′-cGAMP in the HgmTad2-3′,3′-cGAMP structure. 2Fo-Fc electron density of 3′,3′-cGAMP is shown and contoured at 1 σ. e, Structural superposition between HgmTad2-3′,3′-cGAMP and HgmTad2-cGG. 3′,3′-cGAMP and cGG bind at the same position. f, The binding pocket of cGG in the HgmTad2-cGG structure. 2Fo-Fc electron density of cGG is shown and contoured at 1 σ. g, Structural superposition among HgmTad2 in the apo form (two types of conformations) and cGG-bound form. HgmTad2 in the apo form is coloured orange and green for two types of conformations, respectively. HgmTad2 in the cGG-bound form is coloured light magenta and pink for the two protomers. h, Closer view of the binding of the adenine base of 3′,3′-cGAMP in the binding pocket of HgmTad2. i, Native PAGE showed the binding of HgmTad2 mutants to 3′,3′-cGAMP. For gel source date of c and i, see Supplementary Fig. 1.
Extended Data Fig. 10 HgmTad2 has anti-Thoeris activity, but lacks anti-CBASS and anti-CRISPR-Cas activity in vivo.
a,b CapV enzyme activity in the presence of 3′,3′-cGAMP and resorufin butyrate. The enzyme activity rate was measured by the accumulation rate of fluorescence units (FUs) per second. HgmTad2 (8 µM) was incubated with 3′,3′-cGAMP (0.8 µM) for 10 min and then proteinase K (0.708 mg/mL) was added to release the nucleotide from the HgmTad2 protein. Filtered nucleotide products were used for the CapV activity assay. Data are mean ± SD (n = 3 independent experiments). P-value: ****p < 0.0001. **p = 0.0012. c, Plaque assays with 10-fold dilutions of phage F10 to test the activity of SPO1Tad2 and HgmTad2 against SIR2 containing Thoeris system in vivo. Acb2/Tad2 expressed from p30T plasmid. d, Plaque assays with 10-fold dilutions of PaMx41∆acb2 to test the activity of SPO1Tad2 and HgmTad2 against Type II-AGA CBASS in vivo. e, In vitro DNA cleavage with SpyCas9 (100 nM), sgRNA (150 nM), substrate DNA (10 nM), and AcrIIA11/Tad2 proteins (10 µM). Cleavage produce presence indicates no inhibitor activity. For gel source date, see Supplementary Fig. 1. f, Plaque assays with 10-fold dilutions of phage JBD30 to test the activity of HgmTad2 against CRISPR-Cas9 system in P. aeruginosa.
Extended Data Fig. 11 GPBTad2 is active against Thoeris SIR2 and CBASS II-AGA in vivo.
a, Plaque assays to test the activity of GPBTad2 against SIR2 containing Thoeris system in vivo. Organization of P. aeruginosa Pa231 Thoeris operon shown. F10 phage was spotted in 10-fold serial dilutions on a lawn of P. aeruginosa cells (PAO1) expressing Pa231 Thoeris operon genes (PAO1:Tn7 Thoeris SIR2), or cells without the system (PAO1:Tn7 empty), electroporated with pHERD30T plasmids carrying Acb2 and GPBTad2 genes or empty vector. b, Plaque assays to test the activity of GPBTad2 against Type II-AGA CBASS in vivo. Organization of the P. aeruginosa Pa011 CBASS II-AGA operon shown. PaMx41∆acb2 was spotted in 10-fold serial dilutions on a lawn of Pa011 cells with deletion of CBASS operon (Pa011ΔCBASS II-A) or Pa011 wild type cells (Pa011 wt), electroporated with pHERD30T plasmids carrying Acb2 and GPBTad2 genes or empty vector.
Supplementary information
Supplementary Information
Supplementary Notes, Supplementary Discussion and Supplementary Figs 1–14. Supplementary Notes: structural analysis of the complex of Tad1/Tad2 and cyclic oligonucleotides, as well as functional experiments showing that Tad1/Tad2 inhibits the Thoeris system and CBASS system in vivo. Supplementary Discussion: a further summary of the ability of HgmTad2 to bind to cGG. Supplementary Figure: the original images of all gel images in the text, the original data of ITC assays, the phylogenetic tree analysis of Tad1/Tad2 and other experimental results that are not shown in the main text and Extended Data.
Supplementary Table 1
Bacterial hosts containing CD-NTase family infected by Tad-encoding phages.
Supplementary Table 2
Peptides identified by MS.
Supplementary Table 3
Protein sequences used in the study.
Source data
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Li, D., Xiao, Y., Fedorova, I. et al. Single phage proteins sequester signals from TIR and cGAS-like enzymes. Nature 635, 719–727 (2024). https://doi.org/10.1038/s41586-024-08122-4
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DOI: https://doi.org/10.1038/s41586-024-08122-4
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