A phage-encoded sponge protein suppresses bacterial TIR-Caspase immune signaling

Domain: neuroscience
Published: 2026-04-13
Editorial outcome: major_revision

Abstract
Triggering immune response through generation of signal molecules is a common immune strategy across all domains of life. In the bacterial type IV Thoeris anti-phage system, a Toll/interleukin-1 receptor (TIR)-domain protein produces adenosine 5’-diphosphate-cyclo[N7:1’’]-ribose (N7-cADPR) as the immune signal to activate a Caspase-like effector. Here, we identified an inhibitor of type IV Thoeris through a phage mating assay that allows a sensitive phage to acquire anti-defense genes from related resistant phages. The inhibitor (hereafter TadIV-1) functions as a sponge that sequesters the N7-cADPR signal to inhibit Caspase activation. Structural analyses of TadIV-1 indicate a distinctive signal binding mechanism, wherein the binding pocket comprises its N-terminal flexible loop. In addition, phages lacking TadIV-1 can escape type IV Thoeris sensing through mutation in the capsid vertex protein. Collectively, this work expands the phage anti-defense arsenal with a unique immune signal sequestration mechanism and provides insights into phage invasion recognition mechanism of type IV Thoeris.

Introduction
The constant arms race between bacteria and phages has driven the evolution of extremely diverse and sophisticated defense systems 1-9. In turn, phages encode a wealth of anti-defense strategies to escape the host immunity and enable their own survival and proliferation 10-12. Typically, phage-encoded anti-defense mechanisms target defense proteins and disrupt their immune functions, such as many anti-CRISPR proteins 13,14. Alternatively, to inhibit defense systems that produce immune-activating signaling molecules, including type III CRISPR-Cas 15,16, CBASS 17,18, Pycsar 19, Thoeris 20, and Kongming 21, phage proteins can sequester or cleave the immune signals, or deplete their precursor molecules 21-28.

Thoeris exploits a TIR (Toll/interleukin-1 receptor) domain, which is also a key component of immune mechanisms in animals and plants, to trigger immune signaling 29,30. The TIR protein, upon detection of phage invasion, processes nicotinamide adenine dinucleotide (NAD+) into ADP-ribose (ADPR) derivatives as immune signals, which then activate downstream effectors, such as NAD+-consuming enzymes, to trigger cell dormancy or cell death and abort phage replication 20,31. Therefore, anti-defense proteins may target either the immune signal or the protein components to escape Thoeris immunity. For example, Thoeris anti-defense 1 (Tad1) and Tad2 act as sponge proteins and sequester the 1”-3’ glycocyclic ADPR (3’-cADPR) signal to inhibit type I Thoeris 23,27,28. In addition, a clade of Tad2 homologues sequesters the type II Thoeris signal, histidine-ADPR 31. Notably, these anti-Thoeris sponge proteins can form distinct binding sites and simultaneously sequester multiple immune signals 27. By comparison, Tad3-Tad8 directly bind to TIR domains or Thoeris effectors to inhibit Thoeris immunity 32. In addition, TadIII-1 suppresses type III Thoeris immunity, yet the mechanism of action is unknown 33.

Most recently, a type IV Thoeris system that generates the unique signal adenosine 5’-diphosphate-cyclo[N7:1’’]-ribose (N7-cADPR) to activate a Caspase-like effector was discovered 34. However, how phages escape type IV Thoeris immune signaling remains largely unclear. In this study, we found that whereas T6 is sensitive to type IV Thoeris 34, its closely related phage, T4, can escape the immunity. Through phage mating between T6 and T4, we identified that T4 encodes an anti-type IV Thoeris protein (TadIV-1) that is responsible for the immune evasion. TadIV-1 counteracts defense by sequestering N7-cADPR to inhibit Caspase activation. Our structural analyses indicate that TadIV-1 assembles into a homodimer with each N-terminal flexible region constituting an N7-cADPR binding pocket. Together, our findings expand the repertoire of phage sponge proteins and uncover a distinctive signal-binding mechanism.

Results
Identification of an anti-defense protein inhibiting the type IV Thoeris system

Comparative genomics of closely related phages that show distinct sensitivity to bacterial immune systems has led to the discovery of anti-defense proteins 23,28. To identify an anti-defense protein of type IV Thoeris, we challenged the system with a collection of phages in our lab. This revealed that it strongly protected host cells against T2 and T6, but not T4 (Fig. 1A). The three T-even phages are closely related and share substantial genomic regions with high sequence homology (Fig. S1A). These observations suggest that the unique genes of T4 may encode an anti-type IV Thoeris factor; alternatively T2 and T6 may contain specific triggers for the defense system. To identify the potential determinant, we set up a phage mating assay that allows T6 to obtain or exchange genomic regions from T4, and thus to escape type IV Thoeris 35. In the phage mating assay, we constructed an artificial CRISPR-Cas13 system to counter-select T4 via T4-specific spacers (Fig. 1B) 36. Analysis of the immunity of the CRISPR-Cas13 system confirms that it only targets T4 (Fig. S1B). We then constructed a strain containing both Cas13 and type IV Thoeris systems, and as expected, neither T4 nor T6 could form plaques on the strain (Fig. 1C). In addition, co-infection of T4 and T6 did not generate plaques (Fig. 1C). Thus, we co-infected a sensitive host with both T4 and T6, collected their progenies, which may contain hybrids, and replicated the progenies using the double defense strain (Fig. 1B). Then, the resulting phage hybrids were analyzed using plaque forming assay with the double defense strain. This resulted in clear plaques, indicative of hybrids that could escape both immune systems (Fig. 1C).

Next, we isolated and sequenced 9 hybrid phages. Analysis of their genomes revealed that they are T6 derivatives with genomic segments from T4 (Fig. 1D). Notably, all 9 hybrid phages carry a ~3 kb T4 segment, suggesting that this segment may be responsible for the escape from type IV Thoeris. We then divided this segment into two parts to test whether one specific region was responsible for inhibiting type IV Thoeris (Fig. 1D). While construction of the expression plasmid of Part II failed (data not shown), likely due to toxicity of the encoded proteins, Part I enabled T6 to escape type IV Thoeris (Fig. 1E). Analysis of each gene in Part I revealed that co-expression of the vs.3 gene with type IV Thoeris impaired the immunity (Fig. S1C). Moreover, deletion of vs.3 from Part I abolished immune suppression (Fig. S1D), indicating that vs.3 encodes an anti-type IV Thoeris protein. When expressed from an araBAD promoter, Vs.3 completely inhibited type IV Thoeris immunity (Fig. 1F). Furthermore, deletion of vs.3 from T4 rendered the phage sensitive to type IV Thoeris, whereas complementation of the protein restored the infectivity, suggesting that Vs.3 is the sole anti-type IV Thoeris protein of T4 (Fig. 1G). Collectively, we identified T4 Vs.3 as the anti-defense protein against type IV Thoeris, and thus designated it TadIV-1 (type IV Thoeris anti-defense protein 1).

TadIV-1 inhibits type IV Thoeris by sequestering N7-cADPR

Type IV Thoeris uses the N7-cADPR signal to trigger an immune response 34, and thus TadIV-1 may target the signal to inhibit the immunity. To reveal the mechanism of TadIV-1, we set up a PsCaspase reporter assay as described previously 34, wherein PsCaspase can be activated by filtered cell lysate of infected TIR-expressing cells, which contains N7-cADPR, and degrade a synthetic peptide substrate, generating fluorescence (Fig. 2A). When TadIV-1 was co-expressed with TIR, PsCaspase activation was diminished (Fig. 2B), suggesting that TadIV-1 either prevents N7-cADPR synthesis or removes the signal from the cell lysate.

Next, we purified TadIV-1 and incubated it with N7-cADPR. The incubation also abolished PsCaspase activation (Fig. 2C), indicating that TadIV-1 directly targets N7-cADPR. In addition, the inhibition was dependent on TadIV-1 concentrations, but not the incubation time (Fig. 2C), suggesting that TadIV-1 binds to N7-cADPR rather than degrades it. Moreover, liquid chromatography (LC) analysis showed that incubation of TadIV-1 sequestered the free N7-cADPR, while denaturation of TadIV-1 released N7-cADPR (Fig. 2D). The released N7-cADPR post TadIV-1 incubation still efficiently activated PsCaspase (Fig. 2E), reinforcing that TadIV-1 binds to N7-cADPR rather than degrades it. The affinity between TadIV-1 and N7-cADPR was analyzed by surface plasmon resonance (SPR), which showed a KD of ~216 nM (Fig. 2F). Together, the results indicate TadIV-1 acts as a protein sponge that sequesters N7-cADPR to inhibit type IV Thoeris signaling.

Anti-defense sponge proteins usually bind to different signaling molecules and thus can suppress multiple defense systems 25-27. To analyze whether TadIV-1 also has this ability, we co-expressed TadIV-1 with type I, type II Thoeris systems 20,31, two CBASS systems 37, and Kongming system 21, respectively, and infected the co-expression strains with phages that are sensitive to the defense systems. The results showed that TadIV-1 did not suppress the immunity of type II Thoeris system, Kongming system, or the Escherichia albertii CBASS (Fig. S2A). Although TadIV-1 slightly restored the infectivity of T5 and T6 on the plates containing type I Thoeris and the Yersinia CBASS, respectively (Fig. S2A), in vitro assays failed to detect direct binding of TadIV-1 to the signaling molecules of type I Thoeris or YaCBASS (Fig. S2B). These data suggest that TadIV-1 specifically sequesters N7-cADPR or targets a narrow range of immune signals, in contrast to other sponge proteins.

Sequence features and distribution of TadIV-1

Like most previously described anti-defense proteins, TadIV-1 is a small protein with 92 amino acids lacking a recognizable protein domain. A homology-based search identified 432 TadIV-1 homologs in the INPHARED database and 126 homologs in the NCBI non-redundant protein database. Most TadIV-1 homologues are encoded by phage genomes, except a few lacking genomic annotation (Supplementary Data 4, Fig. 3A). These phages belong to the Straboviridae family and mainly infect bacteria of the Enterobacteriaceae family. Analysis of protein length revealed that they can be classified into two distinct groups, one with the typical protein length as T4 TadIV-1 (~90 a.a) and another significantly shorter group (~70 a.a.) (Fig. 3B). Sequence alignment indicates that the long group is extended at the N-terminal region (Fig. S3). We selected 11 TadIV-1 homologues, covering both the long and short groups, and expressed them using an araBAD promoter in the strain containing type IV Thoeris. Infection of the co-expression strain with a sensitive T4 mutant (vs.3 deletion) revealed that all the long homologues were able to inhibit type IV Thoeris, whereas the short version did not enable phage escape (Fig. 3C, Fig. S3), suggesting that the N-terminal region is essential for the anti-Thoeris function. Notably, T2, although sensitive to type IV Thoeris, also encodes a functional TadIV-1 homologue. The sensitivity of T2 might result from insufficient expression of its TadIV-1 under the experimental conditions.

TadIV-1 is a dimer with flexible binding pockets

To further understand how TadIV-1 sequesters N7-cADPR, we purified a TadIV-1 homolog from Escherichia phage UPEC07 (U07TadIV-1), which was more stable than T4 TadIV-1 in vitro. SEC-MALS (multi-angle light scattering coupled with size exclusion chromatography) analysis indicated that U07TadIV-1 formed a dimer in solution (Figure S4A). Consistently, determination of the X-ray structure of apo U07TadIV-1 (2.56 Å resolution, Table S1, Fig. S5A) revealed that TadIV-1 folds as a two-fold symmetrical homodimer with two anti-parallel helices in each protomer (Fig. 4A). Interestingly, among the full-length 101 residues of UPEC07 TadIV-1, no electron density for the N-terminal 32 residues in each protomer was detected, suggesting its flexible nature (Fig. 4A). The AlphaFold3 (AF3) server 38 also predicted this region as an unfolded structure with low confidence (Fig. S5B), while the remaining part aligned well with the solved structure (Fig. S5C).

Next, we sought to prepare the N7-cADPR-bound TadIV-1 sample. To this end, we mixed purified TadIV-1 with the lysate of T6-infected TIR-expressing cells and then further purified the TadIV-1 protein. The Absorbance 260nm/280nm ratio of the purified TadIV-1 increased from ~0.6 to ~1.5 after incubation with the cell lysate (Fig. S6A). Further, proteolysis of the TadIV-1 sample released molecules that activated PsCaspase (Fig. S6B), suggesting that the TadIV-1 had bound to N7-cADPR. Binding to N7-cADPR did not affect the oligomeric status of TadIV-1 (Fig. S4B). However, despite experimentally solving over one hundred structures of N7-cADPR-bound TadIV-1 under different crystallization conditions, there was still no density for the N-terminal 32 residues in each protomer or the N7-cADPR signal. The remaining 33-101 part of the structure was also the same as the apo structure (Fig. 4B). This suggests that the flexible N-terminal region might be responsible for N7-cADPR binding. Supporting this, a truncated form of TadIV-1 (residues 33-101, Δ2-32) almost lost its ability to inhibit PsCaspase activation in vitro and suppress the immunity in vivo (Fig. 4C-D). This is also consistent with the results that the short homologs of TadIV-1 did not enable phage escape (Fig. 3C). By comparison, a longer truncated form (residues 19-101, Δ2-18) retained its anti-defense ability (Fig. 4C-D), suggesting that the major residues involved in N7-cADPR binding might be in the region ranging from 19 to 32.

To further investigate the binding mode of N7-cADPR, we co-fold TadIV-1 with N7-cADPR and predicted 200 structures using the boltz2 server 39. Interestingly, each prediction suggested that there are two identical binding pockets in a TadIV-1 dimer (Fig. S5D. Additionally, most predictions place N7-cADPR between the N-terminal flexible loop of one protomer and α1 helix of the other protomer (Fig. 4E and S5D). Out of the predictions, we selected out the ones that are consistent with the current finding that the residues 19-32 are implicated in N7-cADPR binding. In these predictions, R18, R19, S21, and S23 from the N-terminal loop and K34 and E58 from the α1 helix directly interact with N7-cADPR (Fig. 4F). Mutational studies of these residues revealed that the mutations impaired the anti-defense activity both in vitro and in vivo (Fig. 4G-H). In particular, combined mutation of S16, R18, R19, S21, S23, N24, K34, and E58 almost completely abolished the anti-defense function, supporting our model that the TadIV-1 dimer binds two N7-cADPR molecules using binding pockets consisting of the N-terminal flexible region. In addition, the TadIV-1 dimer contains a positively charged region, which is close to the N7-cADPR-binding pocket but does not directly interact with N7-cADPR in our models (Fig. 4I, Fig. S5D-F). Mutation of the implicated basic residues (K57, K60, H83) also impaired the anti-defense activity (Fig. 4G-H), yet the specific function of these residues remains unknown.

To investigate whether T4TadIV-1 exploits a similar N7-cADPR binding mechanism, we also predicted the structure of N7-cADPR-bound T4TadIV-1. Within the predicted models, a subset of them also shows that T4TadIV-1 binds to N7-cADPR with its N-terminal flexible region (Fig. S7A-B). Mutational analysis of the conserved residues, R17, R18 and K24, counterparts of R18, R19 and K34 of U07TadIV-1, respectively, indicated their important function in N7-cADPR binding (Fig. S7C-D). Together, the data indicate that T4 and U07 TadIV-1 homologs share a conserved signal-sequestering mechanism.

TadIV-1 is structurally distinct from all known sponge proteins

Up to now, six families of phage proteins have been identified as sponge proteins, including Tad1, Tad2, Acb2, Acb4, sequestin and lockin (Fig. S8A-F) 23,25,28,40,41. All of these proteins are tetramers or hexamers, except sequestin, which is a dimer or tetramer. More importantly, all known sponge proteins form stable binding pockets for nucleotide signaling molecules, either resolved in crystal structures or predicted with high confidence (Fig. S8A-F). However, TadIV-1 shows a markedly different pocket, which comprises a highly flexible N-terminal loop. Moreover, TadIV-1 appears to display a binding specificity for N7-cADPR (Fig. S2), while all the other sponge proteins except sequestin have been reported to sequester at least two types of signaling molecules 25-27. Interestingly, two-helical structures are also found in two other anti-defense sponges: Acb2 and sequestin (Fig. S8G). However, TadIV-1 is also markedly different from the two sponges in both structure and ligand binding. Sequestin is a dimer presenting a standard back-to-back arrangement, with two ligands sandwiched between the two protomers along the long axis (Fig. S8E). However, two TadIV-1 protomers mainly interact with each other with the terminal region of the two-helical structure but display free middle regions (Fig. S8G). Moreover, in TadIV-1 the binding pockets are mainly formed by the N-terminal flexible loops (Fig. 4E-F). The helical region of Acb2 is much longer than that of sequestin and TadIV-1, and Acb2 also has a kink in the C-terminal part. Together, TadIV-1 represents a novel family of sponge proteins with unique structural features.

Mutation of the capsid vertex protein enables T6 to escape type IV Thoeris

Experimental phage evolution can provide insights into the bacteria-phage arms race by selecting phage mutants that evade immunity, through mutations of components that activate defense systems, acquisition of anti-defense functions, or other strategies 25,42-47. We sought to isolate T6 mutants that escape type IV Thoeris. Since plating of T6 on cells carrying type IV Thoeris did not yield any plaques, we used the Appelmans protocol to evolve T6 43, and obtained phage escapers resistant to type IV Thoeris (Fig. 5A, Fig. S9A). Genomic sequencing indicated that all escapers have the same mutation (A380V) within the gene encoding a capsid vertex protein (Gp224), in line with that many defense systems sense phage capsid proteins as a trigger 19,42,44,45. Next, we exploited the PsCaspase reporter assay to analyze the generation of the immune signal during infection by the escaper (Fig. 5B). This indicates that infection by T6 escaper failed to trigger N7-cADPR production (Fig. 5B). To analyze whether the capsid vertex protein alone activates type IV Thoeris, we co-expressed it under a Ptet promoter with type IV Thoeris. However, the co-expression did not induce any cytotoxicity (Fig. 5C), suggesting that activation of the immune signaling necessitates other phage components. We were also unable to detect direct interaction between Gp224 and TIR (Fig. S9B). Then, the Gp224 and type IV Thoeris co-expression strain was challenged by wild-type T6 and T6 escaper. The results showed that expression of Gp224 restored the immunity against T6 escaper (Fig. 5D). Overall, the data indicate that the capsid vertex protein is essential for activation of type IV Thoeris, while other phage components are likely required.

Methods
Bacterial strains and phages
Escherichia coli strain DH5α was used for plasmid construction, BL21(DE3) for protein expression and phage amplification, and K-12 MG1655 strain for phage challenge assay and generation of signaling molecule. DH5α and BL21(DE3) were grown in Lysogeny Broth (LB) medium or LB agar (LBA, 1.5% agar (w/v)) plates at 37oC. MG1655 was grown in MMB medium (LB+0.1 mM MnCl + 5 mM MgCl + 5 mM CaCl , with or without 0.5% agar (w/v)) at 37oC or 30oC. If applicable, the media were supplemented with ampicillin (100 μg·ml-1) or kanamycin (50 μg·ml-1) or streptomycin (50 μg·ml-1). Phages T2, T4, T5 and T6 used in this study were amplified in BL21(DE3) liquid culture. The infected cultures were centrifuged at 6,000 g for 10 min and the supernatants were filtered through 0.22 μm membrane filters (Millipore, SLGPM33RS). The resulting phage stocks were stored at 4oC.

Plasmid and strain construction
The proteins expressed in this study and their sequences are shown in Supplementary Data 1. The plasmid vectors used in this study and the constructed plasmids are listed in Supplementary Data 2, and primers used in this study are summarized in Supplementary Data 3.
To analyze the anti-phage function of type IV Thoeris system, a representative member from Escherichia coli (EcThoeris) was codon optimized, sythesized by GENERAL BIOL, and cloned into the pBAD/His_A vector together with its native promoter. In addition, Type IV Thoeris from Pseudomonas sp. 1-7 (PsThoeris), Type I Thoeris from Bacillus cereus, Type II Thoeris from Bacillus velezensis, CBASS from Escherichia albertii (EcCBASS) and CBASS from Yersinia aleksiciae (YaCBASS) were sythesized by GENERAL BIOL, and cloned into the pBAD/His_A vector under an araBAD promoter. To construct inducible expression EcThoeris, the EcTIR and EcCaspase encoding genes were amplified using primers pBAD-EcTIR-F and pBAD-EcCaspase-R, and then inserted into pBAD/His_A vector vector between the NcoI and HindIII sites. To construct the strain that generates N7-cADPR upon phage infection, the EcTIR encoding gene was amplified using primers pUC19-EcTIR-F and pUC19-EcTIR-R, and then inserted into pUC19 vector between the Sph1 and EcoR1 sites; the L-arabinose-induced EcTIR expression plamid was also constructed using the pBAD/His_A vector. PsCaspase was amplified using primers pBAD-PsCaspase-F and pBAD-PsCaspase-R, and then cloned into the pBAD/His_A vector under araBAD promoter for the protease reporter assay.
To determine the anti-type IV Thoeris gene in the exchanged region in phage mating assay, the Part I, Part II, and Part IΔvs3 regions were amplified from the T4 genome using primers listed in Supplementary Data 3. Attempts to insert the fragments into the pCDF vector only generated plasmids pCDF-Part I and pCDF-Part I∆vs.3, whereas construction of the Part II expression plasmid was not successful. pCDF-Part I was used as template for amplification of a linear vector carry the promoter of vs.8, and then the linear vector was assembled with gene sequence of each gene from the Part I region to construct the plasmids expressing Vs.3-Vs.8 respectively, such that expression of these genes is deriven from the promoter of vs.8.
To co-express TadIV-1 with type IV Thoeris, we firstly constructed pCDFara by replacing the T7 promoter with the araBAD promoter derived from pBAD/His_A, and then amplified the TadIV-1 gene from the T4 genome using primers pCDFara-vs3-F and pCDFara-vs3-R, and inserted it into the pCDFara vector between NcoI and XhoI sites. The expression plasmids of TadIV-1 homologues were constructed in a similar procedure using pCDFara as the backbone vector, except that the genes were synthesized by GENERAL BIOL.
To purify T4 TadIV-1 and the homologue protein from Escherichia phage UPEC07 (U07TadIV-1), the protein-encoding genes were amplified by PCR using the plasmids carrying them as template, and cloned into a modified pET28a vector and native pET28a vector respectively, such that T4 TadIV-1 contains a His6-sumo tag, while U07TadIV-1 contains a His6 tag. To express U07TadIV-1 mutants, the plasmid pET28a-U07TadIV-1 was used as template for amplification of linear plasmids carrying the desired mutations with primers listed in Supplementary Data 3, followed by circularization of the linear plasmids.
To construct the CRISPR-Cas13 system for phage mating, a spacer sequence specifically targeting the T4 beta-glucosyltransferase gene was generated by annealing of primers T4-bgt-spacer-F and T4-bgt-spacer-R, and inserted into the pBA559 vector as described previously 36, generating pBA559-bgt.
To construct the CRISPR-Cas13 system for deletion of T4 vs.3, a spacer sequence specifically targeting T4 vs.3 was generated by annealing of primers T4-vs3-spacer-F and T4-vs3-spacer-R, and inserted into the pBA559 vector, generating pBA559-Vs.3. Meanwhile, homologous recombination template for vs.3 deletion was assembled by overlapping PCR of both upstream and downstream ~500 bp regions flanking the T4 vs.3 gene, and cloned into linearized pUC19 to generate pUC19-Vs.3.
To construct inducible expression plasmids of Gp224 and Gp224A380V, T6 gp224 gene with its native promoter were amplified from ancestral and evolved T6 genomes using primers T6-npgp224-F and T6-npgp224-R, and then inserted into the pCDF vector, generating plasmids pCDF-gp224 and pCDF-gp224A380V. To analyze the cell viability of cells co-expressing type IV Thoeris and T6 Gp224, we firstly constructed pCDFtet by replacing the T7 promoter with the Ptet promoter derived from pBA559, then amplified the gp224 gene from T6 genome using primers T6-gp224-F and T6-gp224-R, and inserted it into the pCDFtet vector between XhoI and PacI sites.
To purify EcTIR, the protein-encoding EcTIR gene was amplified using primers ectir--nde1-F53 and strep-ectir-soeR57, and cloned into the pCDFDuet-1 vector, such that EcTIR contains a strep tag. To purify Gp224, T6 gp224 gene was amplified using primers gp244-F56_Nde1 and gp244-R55_Not1, and then inserted into the pET30a vector between NdeI and NotI sites, such that Gp224 contains a His6 tag.
Cloning was performed using ClonExpress II One Step Cloning Kit (Vazyme, C112-02) or restriction-ligation cloning method. All plasmids were verified by Sanger sequencing (Wuhan Tianyi Huayu Gene Technology, China). The plasmids were transferred into corresponding E. coli strains to construct the experimental strains used for the assays below.

Protein expression and purification
Proteins were expressed in E.coli BL21(DE3) strain. Overnight cultures were diluted 1:100 in LB mediun and grown to an optical density (OD600) of ~1.0 at 37oC. Then the cultures were induced by 0.2 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After induced at 16oC for 16 h, the cells were centrifugated at 5,000 g for 10 min and the pellets were resupended in lysis buffer (20 mM Tris-HCl pH7.5, 500 mM NaCl, 30 mM imidazole). Cells were lysed by high-pressure and clarified by centrifugation at 10,000 g for 1 h. Lysates were filtered through 0.22 μm membrane filters (Millipore, SLGPM33RS) and passed over 2 ml of Ni-NTA resin (Cytiva, Sweden). The resin was washed with lysis buffer and wash buffer (20 mM Tris-HCl pH7.5, 500 mM NaCl, 60 mM imidazole), and protein was eluted with elution buffer (20 mM Tris-HCl pH7.5, 500 mM NaCl, 300 mM imidazole). Samples were concentrated with 10-kDa MWCO centrifugal filter units (Millipore, UFC901008) and further purified using a Superdex-200 increase 10/300 GL (Cityva) column pre-equilibrated with bufferD (20 mM Tris-HCl pH7.5, 500 mM NaCl). The eluted proteins were concentrated, aliquoted, flash frozen in liquid nitrogen and stored at -80oC before use. Purity of the proteins were analysed by SDS-PAGE. For T4TadIV-1, the fusion protein with His6-sumo tag was digested with Ulp on the Ni-NTA resin at 16oC for 3 h after removing contaminant proteins with wash buffer. The samples of TadIV-1 and its mutants were checked with SDS-PAGE and shown in Fig. S10.
To purify Strep-tagged TIR, the cells expressing EcTIR were resupended in bufferW (20 mM Tris-HCl pH8.0, 250 mM NaCl) and passed over 2 ml of Strep-Tactin resins (IBA, Germany). The resin was washed with bufferW and eluted with BXT buffer (20 mM Tris-HCl pH8.0, 250 mM NaCl, 50 mM biotin). Samples were concentrated with 10-kDa MWCO centrifugal filter units and further purified using a HiTrapTM desalting (Cityva) column pre-equilibrated with bufferW. The eluted proteins were used to pull-down assay.

Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS)
Static light scattering experiments of U07TadIV-1 and U07TadIV-1+N7-cADPR were performed in 25 mM Tris-HCl (pH 7.4), 500 mM NaCl, using a GE Healthcare Superdex 200 Increase 10/300 GL size-exclusion column coupled to a Wyatt DAWN HELEOS laser photometer and a Wyatt Optilab T-rEX differential refractometer. Data were analyzed using Wyatt ASTRA 7.3.2 software.

Pull-down assay
Strep-tagged EcTIR and His6-tagged Gp224 were incubated on ice for 1 h at a final molar ratio of 1:1. For the controls, the proteins were replaced with an equal volume of bufferW. Then, the samples were incubated with 20 μl of Strep-Tactin resins pre-equilibrated with bufferW on ice for 1 h. The resins were washed with bufferW for 3 times and eluted with BXT buffer. All samples were analysed using 4-20% FuturePAGE™ (ACE, China) and stained with Coomassie Brilliant Blue.

Plaque assays
E. coli MG1655 cells that carried indicated plasmids were subjected to plaque forming assay. The cells were grown in MMB medium at 37oC overnight. Then, 100 μl of bacterial culture was mixed with 6 ml of melted MMB 0.5% agar, poured on MMB 1.5% agar plates and incubated at 37oC for 2 h. To induce the expression of TadIV-1 or TadIV-1 homologues, 0.1% L-arabinose was added to the medium; for U07TadIV-1 and its mutants, 0.005% L-arabinose was used. Then, 3 μl of ten-fold serial dilutions of phages was dropped on the bacterial layer. Plates were incubated overnight at 37oC. Plaque-forming units (PFU) were determined by counting the derived plaques, and lysate titer was determined by calculating PFU·ml-1. If applicable, antibiotics were supplemented into the media and plates for plasmid maintenance.

Phage-infection dynamics in liquid medium
Overnight cultures of E. coli MG1655 cells that carried indicated plasmids were diluted 1:100 in MMB medium supplemented with 0.1% L-arabinose and grown to OD600 of ~0.2 at 37oC. Then, 200 μl of the cultures were transferred into wells of the 96-well plate, and 5 μl of the phages were added into the cultures for a final MOI=0.5. The uninfected cultures were added with 5 μl of the MMB medium. The plate was incubated in a Spark multimode reader (Tecan, Switzerland) at 37oC and OD600 was measured every 5 min with shaking at 200 rpm.

Cell viability
Overnight cultures of E. coli MG1655 cells that carried indicated plasmids were diluted 1:100 in MMB medium and grown to OD600 of ~0.2 at 37oC. Then, aTc was added to a final concentration of 80 ng·μl⁻¹, and the cultures were incubated at 37°C for 2 hours. Serial dilutions were performed, and 5 μl of each solution was dropped on the MMB 1.5% agar plates and incubated at 37oC overnight.

Phage mating assay
A mixture of T4 and T6, each at a titer of 10⁷ PFU·ml-1, was used to infect E. coli MG1655 culture (OD₆₀₀ = 0.2). After incubated at 37oC for 4 h, supernatant of the culture was collected by centrifugation at 10,000 g for 10 min at room temperature. Then, the supernant was used to infect the double defense E. coli MG1655 strain carrying pBA559-bgt and pBAD/His_A-Thoeris, and the supernatant containing hybrid phages was prepared as described above. The supernatant was analyzed by plaque assays to assess its infectivity to the double defense strain.
To isolate single hybrid phage strains, the supernatant was mixed with the double defense cells for 10 min at room temperature, followed by mixed with 6 ml of premelted 0.5% MMB agar and poured onto plates containing 1.5% MMB bottom agar. The double-layer plates were incubated overnight at 37oC and the resulting single plaques were picked into 20 μl of SM buffer (50 mM Tris-HCl pH 7.4, 100 mM MgCl₂, 10 mM NaCl) for further analysis.

Sequencing, assembly and comparison of phage genomes
The supernatant of infected cultures with high-titer phage particles (>10⁷ PFU·ml⁻¹) was used for DNA extraction. A total of 1 ml of the supernatant was treated with 3 μl DNase (Thermo Fisher, EN0521) and 3 μl RNase (Thermo Fisher, EN0531) at 37oC for 1-2 h to remove contaminating bacterial nucleic acids. Following protease K (Thermo Fisher, EO0491) treatment to disrupt the phage capsid, viral DNA was extracted using the TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, 9766) according to the manufacturer’s instructions. DNA samples were submitted to Anoroad Gene Technology for Illumina sequencing. Read pairs were quality-filtered and merged using Fastp v.0.23.4 52, and de novo genome assembly was performed with Unicycler v.0.5.1 53. Assembled hybrid phage genomes were aligned and visualized with SnapGene v.6.0.2. Each hybrid genome was compared to the reference genomes of phages T4 and T6 to identify inserted or deleted regions relative to T6, and to determine the genes contained within these regions.
Comparison of T2, T4 and T6 genomes was performed with CAGECAT webserver 54.

Knock-out of TadIV-1 from phage T4
To generate T4 phage mutant carrying a deletion of vs.3 (T4Δvs.3), 3 μl of wild-type T4 phage (>10⁷ PFU·ml⁻¹) was added to 2 ml of host cells (OD600 = 0.2) carrying the donor plasmid pUC19-Vs.3 and incubated overnight at 37oC. Cultures were centrifuged at 10,000 g for 10 min, and the supernatant containing phages was collected. Homologous recombination in donor-containing host cells led to replacement of the target sequence in a subset of phage genomes. Subsequently, 3 μl of the resulting phage lysate was mixed with 2 ml of host cells (OD600 = 0.2) harboring pBA559-Vs.3, and anhydrotetracycline (aTc) was added to a final concentration of 80 ng·ml-1 to induce Cas13a expression. The mixture was incubated overnight at 37oC to enrich T4Δvs.3 phage. Enriched phages were serially diluted and spotted onto double-layer plates containing the pBA559-Vs.3 strain supplemented with aTc to assess knockout efficiency. Individual plaques were then picked and resuspended in 20–30 μl of SM buffer for subsequent PCR verification of the deletion, and all confirmed knockout isolates were validated by PCR and sequencing.

Sequence analysis and phylogenetic tree construction
TadIV-1 homologs were identified in the INPHARED database (released on 1 Mar 2024) using HMMER v3.4’s jackhmmer with an E-value threshold of 1e-3, with the T4 TadIV-1 sequence as query. Experimentally tested homologs retrieved from the NCBI non-redundant protein database were additionally incorporated into the INPHARED homologs dataset. Redundant sequences were removed using CD-HIT v4.8.1 with a sequence identity threshold of 100% and a length difference cutoff of 100%. Multiple sequence alignment was performed with ENDscript server 55. Phylogenetic trees were constructed with FastTree using the WAG evolutionary model and gamma-distributed site rates, and visualized with the Python package etetoolkit.

Preparation of metabolite fractions
The published procedure was used to prepare metabolite fractions containing N7-cADPR with modifications 34. Specifically, an overnight culture of E. coli MG1655 cells carrying EcTIR was diluted 1:100 in 50 ml MMB supplemented with 500 μM IPTG, and incubated at 37oC with shaking (180 rpm) until an OD600 of 0.4~0.5. T6 phage was added to the culture up to an MOI of 3. The infected culture was incubated at 37oC with shaking (160 rpm), and the cells were collected at 30 min post-infection. The pellet was resupended in 500 μl bufferD and lysed by sonication. The cell lysate was centrifuged at 13,000 g for 10 min and the supernatant was passed through 3-kDa MWCO centrifugal filter units (Millipore, UFC500324) by centrifugation for 30 min at 13,000 g at 4oC. The filtered cell lysate was flash frozen in liquid nitrogen and stored at -80oC before use.
To analyze the anti-defense mechanism of TadIV-1 in vivo, E. coli MG1655 cells co-expressing EcTIR and TadIV-1 were diluted 1:100 in 50 ml MMB supplemented with 500 μM IPTG and 0.2% L-arabinose. Then, metabolite fractions were prepared as described above. Metabolite fractions from cells carrying the empty vectors pUC19 and pCDFara were also analyzed as negative control.
To analyze whether the T6 escaper triggers production of N7-cADPR, E. coli MG1655 cells carrying EcTIR with an araBAD promoter was diluted 1:100 in 150 ml MMB supplemented with 0.2% L-arabinose, then incubated at 37oC with shaking (160 rpm) until an OD600 of 0.4~0.5. Fifty ml of culture was removed as the control sample without infection. The remaining 100 ml was divided equally and infected with wild type or escaper T6 at an MOI of 3, respectively. After 30 min’s incubation, metabolite fractions were prepared as described above.

Peptide cleavage reporter assays
We firstly produced PsCaspase cell lysate as described previously 34. Briefly, the overnight E. coli MG1655 cells expressing PsCaspase was diluted 1:100 in 50 ml of LB and incubated at 37oC with shaking (180 rpm). After 2 h, 0.2% L-arabinose was added to the culture and further incubated until OD600 reached 0.8. The bacterial culture was collected by centrifuged at 6,000 g for 10 min. The pellet was resuspended in 500 μl of 1X Phosphate-Buffered Saline (PBS) pH7.4 (Biosharp, BL302A) and lysed by sonication. The cell lysate was centrifuged at 13,000 g for 10 min. Supernatant was aliquoted, flash frozen in liquid nitrogen and stored at -80oC before use. Then, protease reactions were performed in black 96-well half area plates (Greiner Bio-One, 675076) at 37oC in a 50 μl final reaction volumn. Each reaction mixture containing 25 mM Tris-HCl pH7.4, 100 mM NaCl, 20 μM synthetic 7-amino-4-methylcoumarin (AMC)-conjugated peptide of EEKAR (Genscript) and 2 μl of PsCaspase cell lysates.
Next, 10 μl of metabolites was added to each well immediately before measurement and mixed by pipetting. Then, the plate was immediately incubated in a Spark multimode reader (Tecan, Switzerland) at 37oC and fluorescence intensity was measured every 30 s over 10 min at 341 nm excitation wavelength and 441 nm emission wavelength. Reaction rate was derived from the linear part of the initial reaction.
To analyze the N7-cADPR-sequestering activity of purified TadIV-1, a gradient of concentrations of TadIV-1 were incubated with 250 nM of N7-cADPR (Biolog Life Science Institute) and 1 μl of 10X PBS pH7.4 (Biosharp) in the 10 μl reaction volumn on ice for 5 min, 20 min and 40 min. The mixtures were then analyzed for their ability to activate PsCaspase as described above.
To analyze the N7-cADPR-sequestering activity of TadIV-1 and U07TadIV-1 and their mutants, 2 µl metabolites were incubated with 10 µM TadIV-1 (or mutants) for 10 min. Then, the mixture was supplemented into 50 µl protease reporter assay mixtures containing 25 mM Tris (pH 7.4) and 100 mM NaCl, 40 µM EEKAR substrate and 2 µl PsCaspase lysate. The reactions were performed and the fluorescence was monitored as described above.

HPLC analysis
A reaction mixture containing 100 μM cUUE/UA and 150 μM TadIV-1, or containing 50 μM 3’-cADPR and 100 μM TadIV-1, was incubated in 10 mM Tris pH 7.4 and 100 mM NaCl at 16oC for 4 h. The samples were filtered through a 3 kDa cutoff ultrafiltration tube, and the flow-through fraction was analyzed using an InfinityLab Poroshell 120 AQC18 column (4.6 × 150 mm, 2.7 μm; Agilent). Chromatographic separation was performed at a flow rate of 0.4 ml/min under isocratic elution with 97% 20 mM potassium phosphate buffer (pH 6.0) and 3% acetonitrile. Detection was carried out at 254 nm.
To analyze the N7-cADPR-sequestering activity of TadIV-1, a reaction mixture containing 50 μM N7-cADPR and 100 μM TadIV-1 was incubated in 10 mM Tris pH 7.4 and 100 mM NaCl on ice for 1 h. The reactions were performed and analyzed as described above, except the mobile phase of 10% acetonitrile and the detection UV wavelength at 273 nm.

Release of N7-cADPR by TadIV-1
To examine whether the released molecule from TadIV-1 is able to activate PsCaspase, 10 μM TadIV-1 was pre-incubated with 250 nM N7-cADPR and 1 μl of 10X PBS pH 7.4 in the 10 μl reaction volumn on ice for 10 min. 1 mg·ml-1 of proteinase K (Thermo Fisher, EO0491) was subsequently added and incubated on ice for 1 h. Then the proteinase K was denatured with 80% pre-colded methanol at -80oC for 2 h, and the supernatant after centrifugation (13,000 g, 10 min) was frozen in liquid nitrogen and dried using Freeze Dryer Lichen-10-50C (LICHEN, China). Then, the resulting pellet was dissolved in 10 μl water, and applied for PsCaspase reporter assay.
For HPLC analysis, 100 μM TadIV-1 was pre-incubated with 50 μM N7-cADPR in 10 mM Tris pH 7.4 and 100 mM NaCl. To release N7-cADPR, TadIV-1 was denatured by incubation with 3% SDS.

SPR assay of TadIV-1 and N7-cADPR
The SPR analysis was performed using a Biacore™ 1K (Cytiva, Sweden) at room temperature. TadIV-1 was diluted in immobilization buffer (10 mM sodium acetate, pH 4.5) and immobilized on channels of the carboxymethyldextran-modified (CM5) sensor chip to about 7000 response units (RU). Then, a series of concentrations of N7-cADPR were diluted in HBS-P buffer (Cytiva, BR100368) was injected over the chip at a flow rate of 10 μl/min. The protein-ligand complex was allowed to associate for 100 s and dissociate for 120 s. Data were fit with a steady-state affinity model. The affinity constant (the equilibrium dissociation constant, KD)value was calculated using Biacore evaluation software (Cytiva).

Structural analyses
The protein eluted from the Ni-NTA column was desalted into buffer containing 25 mM Tris pH 7.4 and 200 mM NaCl, and then incubated with the filtered lysate supernatant of TIR-expressing cells. The mixture was concentrated and further purified by size-exclusion chromatography. The fractions containing the target protein were pooled and concentrated to 6 mg·ml-1. Crystallization was carried out at 18oC using the sitting-drop vapor diffusion method with reservoir solution containing 0.1 M ADA pH 6.5 and 1.0 M ammonium sulfate. Before harvesting, the crystals were cryoprotected in the reservoir solution supplemented with 20% (v/v) glycerol and then flash-frozen in liquid nitrogen.
All diffraction data were collected at the SSRF beamlines BL02U1 and BL19U1 and were integrated and scaled using the HKLA2000 package 56. The initial model of U07TadIV-1 was obtained using AlphaFold2 57. All structures were further refined in PHENIX 58 with non-crystallographic symmetry (NCS) and stereochemistry restraints. The final models were obtained after several rounds of refinement. All structural illustrations were generated using PyMOL (https://pymol.org/). Detailed statistics of data collection and structure refinement are summarized in Supplementary Table S1.
Prediction of N7-cADPR-bound TadIV-1 structures was performed with the Boltz2 39. Prediction of the structure of 3’-cADPR-bound Sequestin was performed with Protenix server (https://protenix-server.com/login). The protein sequences are derived from the IMG (IMG Gene Identifier: Ga0209916_1000062263), and the SMILES sequences of 3’-cADPR is obtained from the PubChem. The N7-cADPR structure was drawn using InDraw software (https://www.integle.com/static/indraw) to obtain an SDF file, which was then converted to SMILES format via the website: https://www.novopro.cn/tools/mol2smiles.html.

Modeling of full-length TadIV-1
Structural model of full-length TadIV-1 (UniProt identifier: UPI001BEF590C) was generated using the AlphaFold Server 57 accessed with default parameters on Sept. 12th, 2025.

Isolation and analysis of T6 escapers
To isolate T6 mutants that are resistant to type IV Thoeris, T6 was evolved in liquid media as described previously 43. Briefly, overnight cultures of E. coli MG1655 cells expressing type IV Thoeris were diluted 1:100 in MMB medium and grown to an OD of ~0.3 at 37oC. 100 μl of cultures were transferred into a row of a standard 96-well plate. For the first round of infection, T6 phage was added at a series of MOIs (0, 0.001, 0.01, 0.1, 1, 10). The 96-well plate was incubated at 37oC with shaking (160 rpm). At 4 h post-infection, cultures from cleared wells were collected and centrifuged, and the supernatants were diluted and used to infect a fresh 96-well plate for the next round of evolution. After six rounds of evolution, the cleared wells were pooled and analyzed by plaque assay using the double-layer plate of E. coli MG1655 cells expressing type IV Thoeris. Single phage mutant strains were isolated and sequenced as described for hybrid phages, except that the phage mutants were amplified in E. coli cells expressing type IV Thoeris. In parallel, rounds of infection of E. coli MG1655 cells carrying an empty vector was performed as control, and phages from control evolution were also isolated, amplified and sequenced.
To analyze the mutation sites, the sequencing reads were aligned to the T6 phage reference genome (Accession number: NC_054907) using BWA (Version: 0.7.17-r1188) 59. Single nucleotide polymorphisms (SNPs) were identified with BCFtools (v1.18) 60. Mutations present in both the control and escaped T6 phages were excluded, and silent mutations within protein-coding regions were disregarded.

Data availability
The accession number for the coordinate and structure factor of apo TadIV-1 from Escherichia phage UPEC07 is PDB: 9WIW.
All the data necessary for interpretation are provided in the manuscript or Supplementary Information. Source data are provided with this paper. Sequencing data of phage genomes are available on the NCBI Sequence Read Archive under BioProject ID PRJNA1313588

Code availability
This paper does not report original code.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Chen et al. report identification and characterisation of a phage-encoded sponge protein that inhibits type IV Thoeris defence by binding N7-cADPR. Using a very elegant phage mating strategy and counterselection with Cas13, they identify the gene, which is naturally present in the phage T4 genome. TadIV-1 is shown to bind N7-cADPR both in vitro and to confer resistance specifically to type IV Thoeris (figure 2). Structural studies failed with the full length protein unfortunately, which precluded visualisation of the N7-cADPR bound form, so the authors resorted to modelling and tested predictions by mutagenesis, identifying residues important for function . Finally, the authors identified a role for a capsid vertex protein (Gp224) in activation of type IV Thoeris.\nThis is an interesting paper, but there are major concerns, detailed below, on the purity of N7-cADPR used in all the functional and structural studies, purity and tag-status of the proteins, inconsistencies in the extent of inhibition of pCaspase in the manuscript and quality/confidence of the structural model. Until these are addressed, publication is not supported.\nSpecific points:\n1. According to the methods, the T4 protein was purified with a 6His-SUMO tag on the N-terminus, and this tag was not removed. Is this correct? Given the importance of the N-terminus, it is important to ascertain that an N-terminal tag does not interfere with the structure or function.\n2. While TadIV-1 efficiently reduces pCaspase activity in cell extracts (Figure 2B), the inhibitory action of purified TadIV-1 is much weaker (Figure 2C). Even at very high concentrations of 25 µM, activity is only reduced by about 60%. How can can this be consistent with a dissociation constant of 83 nM (figure 2F)? Surely , nearly complete inhibition should be observed at 1 µM TadIV-1, at >10x KD?\n3. Figure 2E shows that pCaspase activity is reduced by ~90% here. How does this fit with the data in 2C? How much TadIV-1 was added in 2E – it is not stated?\n4. The LC-MS/MS data in figure 2D are of poor quality. Can this be improved? What does the corresponding LC data look like? Supplemental data on the MS analysis would be welcome, in particular to help the reader understand how complex the mixture of nucleotides is in the filtered cell extract. There is presumably still a lot of ATP, other NTPs, NAD etc? Please comment, as this will influence many of the assays in this section of the paper, such as the SPR.\n5. Structural studies unfortunately failed to obtain a full length structure in the absence or presence of N7-cADPR. In its absence, the structural data are incomplete and the modelling takes on a great importance. Although 10 models are shown in figure S4, it is impossible to ascertain whether these match each other, and what the confidence level of each prediction is. The model of the intact protein shown bound to N7-cADPR in figure 4E could be accurate, or not – how can the reader assess this?\n6. Following on from point 5, it is clear that T4 TadIV-1 lacks 8 residues found in the modelled protein from U07. This includes S23 which is shown to be important for binding. Please repeat the modelling with the T4 protein and describe how the two models agree/disagree. If they are not in good agreement, are the models a reflection of the real structure? After all, conserved residues in the N-terminus are always likely to be important for binding, regardless of the structure the protein forms.\n7. In figure 4C, here the WT protein completely inhibits pCaspase. How does this fit with the data in figure 2 where inhibition is only partial? Is this because a different homolog of TadIV-1 was used?\nMinor comments:\n1. Line 165 and 184 space missing\n2. Please add a supplemental figure showing the purified proteins, including the variants show in figure 4.\n3. The SPR data (Figure 2F) have been carried out only once. This needs to be repeated and stats provided.\n4. Line 497 typo “incubaed”.", "reviewer_2": "In the manuscript by Yu et al, the authors identified an inhibitor of type IV Thoeris named TadIV-1. They employed different methods and strategies to demonstrate that TadIV-1 functions as a sponge that sequesters the type IV Thoeris signaling molecule N7-cADPR to inhibit Caspase activation. They determined a crystal structure of apo TadIV-1, but despite numerous attempts, failed to obtain a structure of TadIV-1 bound to N7-cADPR. Structural modeling suggested a ligand binding by a flexible N-terminal loop, which was tested by mutagenesis. The authors claimed that this binding mode is distinct from other characterized sponge proteins, which form deep binding pockets between the protein subunits in oligomers (dimers, tetramers or hexamers). They also found that T6 phage can escape type IV Thoeris by the mutation in the capsid vertex protein gp224. Sponge proteins for type I and II Thoeris are quite well characterized, but information about sponges sequestering the type IV Thoeris signaling molecule is limited. Moreover, despite the numerous studies on Thoeris system, little is known about how the phage infection is recognized by ThsB (TIR) proteins. Therefore data presented in this manuscript is important and relevant to the field.\nMy major concerns:\n1. Common two-helical structure of the sponge proteins. In the manuscript TadVI-1 is distinguished by a distinct binding mode, but its overall structure in comparison to other sponge proteins is not discussed. Two-helical structure is also found in 2 other anti-defense sponges: Acb2 (which was found to bind CBASSS and Thoeris signaling molecules) and Sequestin. It would be more informative in the Fig.S6 to show one subunit of the shown structures in bold color demonstrating structural differences/similarities of the sponge proteins. PDB IDs of the depicted structures should be provided in the Fig. S6 legend. To demonstrate their structural similarity and differences single subunits of Acb2, Sequestin and TadIV-1 next to each other in the same orientation should be shown (e.g. as Fig. S6B) and discussed in the manuscript. Interestingly, in Fig. S4C the N-terminal flexible loop is predicted as a beta-hairpin in a few models. Similarly, the N-terminal hairpin is present in the Acb2 proteins.\n2. Oligomeric state of TadVI-1. Helical sponge proteins form different oligomers, from dimers, tetramers (sequestin) to hexamers (Acb2) (ref 40). Oligomeric form of TadVI-1 in solution is not known. Oligomeric structure in solution of TadIV-1 should be determined (e.g. by SEC-MALS) to demonstrate that it is a dimer in the presence and absence of N7-cADPR. A dimer of U07TadIV-1 is observed in the crystal. Can be other oligomeric structures predicted from the crystal contacts by PISA server?\n3. Model of N7-cADPR binding. The N7-cADPR binding is predicted by boltz2 server. The main message of the manuscript is that TadVI-1 has a distinct signal binding mechanism; since the binding pocket is located not at the interface between subunits, but the binding pocket comprises the TadIV-1 N-terminal flexible loop. However, the N-terminal flexible part of TadIV-1 homologs, that inhibit type IV Thoeris, varies in length (Fig. S3). Only 3 of 8 characterized TdaIV-1 proteins contain longer variant similar to U07TadIV-1, which was characterized in detail. In the remaining 5 homologs this loop region is shorter in 9 aa residues, and it is difficult to imagine, how it could accommodate N7-cADPR in the proposed binding mode. A model of N7-cADPR binding by a TadIV-1 variant with a shorter flexible loop, like T4 TadVI-1 (Fig. S3), should be generated and compared with U07TadVI-1 predicted binding mode. Next, only residues from the flexible N-terminus (except E58) are predicted to make specific contacts with N7-cADPR (Fig. 4F, Fig. S3). Mutational analysis of these N-terminal residues of U07TadIV-1 (mutation of E58 was not performed) demonstrated that the flexible loop is important for the N7-cADPR binding. However, since in the proposed model almost no residues from the helical structure are involved in the binding, there is a possibility that the position of this flexible loop regarding the helical structure may be different from the predicted model. Other sponge proteins contain conserved positive surfaces for binding of cyclic nucleotide molecules (ref 40). The helical structure of U07TadIV-1 also contains a positive surface with the conserved residues K57, K60, H83 and a pocket next to them (in the AF3 model), and it is tempting to speculate that this surface may also be important for the N7-cADPR binding. I suggest mutating the residues of this positive interface to test their importance in N7-cADPR binding. This analysis would strengthen the proposed hypothesis. Electrostatic surface of the predicted U07TadIV-1 bound with N7-cADPR model (shown in Fig. 4E) should be shown, since N7-cADPR contains negative charge which is expected to interact with positive charge on the protein.\n4. In Fig.S2A it is shown that co-expression of TadVI-1 weakened the protection of type I Thoeris by an order of magnitude, similar to YaCBASS. It should be tested if TadIV-1 can bind type I Thoeris signaling molecule 3’cADPR.\n5. Phage escape mutant seems a bit out of topic of this manuscript. The description of gp224 and its possible recognition by Thoeris is very brief. The authors should discuss more about alternative strategies to escape antiviral defense: by acquiring anti-defense proteins of by mutating phage proteins recognized by the defense system.\nMinor comments:\n1. Fig. 2C: the TadIV-1 concentration on X axis is shown in opposite direction (from 25 to 0). More intuitive is to use from 0 to 25.\n2. Fig. S2A: what is EV in this figure?\n3. Fig. 4C Lines in the legend are too thin and colors are too similar: it is difficult to follow.\n4. Lines 225-226 “unprecedented features” sounds too dramatic.\n5. Methods: inconsistence in names (pBADHis_A / pBADHisA), units (PFU.ml-1 / PFU ml-1), etc.\n6. Typos: line 165 “versiondid”, line 184 “hadbound”.", "reviewer_3": "In the manuscript by Chen et. al., the authors identify and characterise a novel phage-encoded sponge protein which enables escape from the type IV Thoeris phage defence system. They produce hybrid phages using hybridisation assays to produce phages comprised of genomic material from the related phages, T4 and T6. Through comparative genomics and EOP assays, the authors identify the TadIV-1 gene as responsible for the escape phenotype. PsCaspase activation assays demonstrate that this inhibition is achieved by sequestration, but not degradation, of the N7c-ADPR Thoeris type IV signalling molecule. The authors go on to solve the structure of the TadIV-1 dimer using x-ray crystallography. Though the putative N7-cADPR binding region is not resolved in the crystal structure, the authors identify regions and residues responsible for N7-cADPR binding through mutational analysis and EOP/PsCaspase assays. Further, the authors also identify a phage trigger of Thoeris type IV through directed evolution of escaper phages.\nOverall, the manuscript is a valuable contribution to the growing work on phage encoded sponge proteins which allow phage escape from a growing repertoire of defence systems which utilise cyclic nucleotide messengers. In particular, the novel binding mechanism and specificity shown by TadIV-1 is of interest. The work is technically sound, the experiments are well planned, and the manuscript is well written. I do however have a few suggestions which I believe would improve the study and ensure that all conclusions therein are thoroughly validated, along with a few minor corrections.\n_____________________________________________________________\nThe authors identify two distinct classes of TadIV-1, long and short. While they extensively test the long version for ability to bind N7-cADPR and to inhibit Thoeris function, only one member representing the short version is tested. Could the authors please test a few additional short versions? This would add support the suggestion of the extended flexible N-terminal region being essential to N7-cADPR binding.\nFollowing on from this point, the authors also carry out a homology-based search for TadIV-1 in phage genome sequences. Are there many cases in which phages encode both short and long versions? If yes, this would support different target molecules/systems. If no, this would suggest some functional redundancy.\nThe authors produce 200 binding models for N7-cADPR. In figure S4C, there does also appear to be a cluster of N7-cADPR bound between the two helices. Are there any conserved ligand poses or binding positions within this cluster? Even if not, this cluster should at least be acknowledged in text.\nIn figure 4, additional mutational analyses should be carried out on identified binding residues from boltz2 ligand modelling. In particular, K34 and E58 account for 5 interactions in panel F. I would recommend the additional mutation combinations based on the interactions shown: K34 and E58 double mutant; S16, R18, R19, S21, S23, S24; and S16, R18, R19, S21, S23, S24, K34, E58. This would validate the ligand binding prediction and potentially show complete abrogation of N7-cADPR binding.\nThe authors also implicated the phage capsid vertex protein in triggering type IV Thoeris. This section should be expended on. In particular, testing for N7-cADPR levels when co-expressing the capsid vertex protein and assessing for direct binding/interactions between the capsid vertex protein and Thoeris components.\nMinor corrections\nIn figure 4, I find the colours in panel C difficult to interpret. I think that a bar chart of endpoint values would be clearer and would be sufficient to convey this information.\nI find the repeated use of the word “unprecedented” to be quite strong for the findings here. Perhaps “novel” or “unique” would be more appropriate.\nPlease state structure resolutions, either in text or in figure descriptions.\nLine 54: Thoeris systems often cause cell growth arrest rather than direct cell death.\nLine 176: Alphafold3.\nLine 410: molten, not “premelted”\nPlease change degrees symbol to the correct degrees symbol throughout.\nVarious spelling and grammar errors throughout methods section, please go through again before resubmission.\nI hope the authors find my comments helpful and I thank them for the opportunity to read their interesting work."}, "author_response": "We thank the reviewer for her/his interest in our work and for the important comments and suggestions.\nSpecific points:\n1. According to the methods, the T4 protein was purified with a 6His-SUMO tag on the N-terminus, and this tag was not removed. Is this correct? Given the importance of the N-terminus, it is important to ascertain that an N-terminal tag does not interfere with the structure or function.\nThanks for the suggestion.\nIndeed, the tag of T4TadIV-1 was not removed in the original manuscript. In the revised manuscript, we obtained the tag-free TadIV-1 through removing of the His-SUMO tag by proteolysis and repeated the in vitro experiments (revised Fig. 2C-F). The tag-free protein showed similar N7-cADPR binding ability to that containing the tag (original Fig. 2C-F). In addition, we analyzed the anti-Thoeris activity of tagged T4TadIV-1 in vivo, and found that the tag did not affect the anti-defense function (Response Figure 1), in line with our in vitro observations.\nResponse Figure 1. The N-terminal tag does not affect the anti-Thoeris function.\n2. While TadIV-1 efficiently reduces pCaspase activity in cell extracts (Figure 2B), the inhibitory action of purified TadIV-1 is much weaker (Figure 2C). Even at very high concentrations of 25 µM, activity is only reduced by about 60%. How can can this be consistent with a dissociation constant of 83 nM (figure 2F)? Surely , nearly complete inhibition should be observed at 1 µM TadIV-1, at >10x KD?\nWe thank the reviewer for raising these important questions regarding the experiments in Figure 2. The discrepancy might be attributed to the differential experimental conditions.\nIn Figure 2B, TadIV-1 was co-expressed with TIR in vivo, while purified TadIV-1 was supplemented into filtered metabolites of TIR-expressing cells in Figure 2C. In addition, the metabolites contained additional contaminat molecules, such as NTPs and NAD+, and the concentration of N7-cADPR in the metabolites was unknown.\nTo address the limitations, we repeated the in vitro experiments using commercial N7-cADPR (C 411, Biolog Life Science Institute) and tag-free TadIV-1, as suggested by the 1st comment. The data (revised Figure 2C) indicate that 10 µM TadIV-1 reduced psCaspase activity by ~95%, consistent with the efficient inhibition in the cell extracts (Figure 2B).\n3. Figure 2E shows that pCaspase activity is reduced by ~90% here. How does this fit with the data in 2C? How much TadIV-1 was added in 2E – it is not stated?\nThanks for the questions.\nAs described in the last Response, we addressed the problem with commercial N7-cADPR. We have clearly indicated the concentration of TadIV-1 used in Figure 2E in the revised legends.\n4. The LC-MS/MS data in figure 2D are of poor quality. Can this be improved? What does the corresponding LC data look like? Supplemental data on the MS analysis would be welcome, in particular to help the reader understand how complex the mixture of nucleotides is in the filtered cell extract. There is presumably still a lot of ATP, other NTPs, NAD etc? Please comment, as this will influence many of the assays in this section of the paper, such as the SPR.\nThanks for the question.\nIndeed, in our original manuscript, N7-cADPR was prepared from TIR-expressing cells and was not pure. In the revised manuscript, we repeated the in vitro assays in Fig. 2, including the LC assay, with commercial N7-cADPR, which avoided the contamination of other nucleotides.\n5. Structural studies unfortunately failed to obtain a full length structure in the absence or presence of N7-cADPR. In its absence, the structural data are incomplete and the modelling takes on a great importance. Although 10 models are shown in figure S4, it is impossible to ascertain whether these match each other, and what the confidence level of each prediction is. The model of the intact protein shown bound to N7-cADPR in figure 4E could be accurate, or not – how can the reader assess this?\nThe reviewer raised a very good point. In the revised Figure S4 (now Fig. S5 in the revised manuscript), we have included the confidence level of each prediction. Out of the ten models, six models showed a similar binding pocket formed by the N-terminal flexible loop, three showed a pocket between the two helices within each protomer, and one showed two pockets in both termini of the antiparallel dimer. We agree with the reviewer that the model shown in Figure 4E could not be completely accurate, however, the readers could still assess its confidence through the results of the mutational studies. Our mutational studies, both in vivo and in vitro, support that the residues shown to be involved in ligand binding in the model indeed participate in N7-cADPR sequestering.\n6. Following on from point 5, it is clear that T4 TadIV-1 lacks 8 residues found in the modelled protein from U07. This includes S23 which is shown to be important for binding. Please repeat the modelling with the T4 protein and describe how the two models agree/disagree. If they are not in good agreement, are the models a reflection of the real structure? After all, conserved residues in the N-terminus are always likely to be important for binding, regardless of the structure the protein forms.\nWe thank the reviewer for this insightful suggestion to predict the structure of N7-cADPR-bound T4TadIV-1. We performed the prediction and the resulting model indicates that T4TadIV-1 also likely binds to N7-cADPR with its N-terminal flexible region, in which the conserved residues R17, R18 and K24 are implicated in the direct interaction (Fig. S7A-B). Further, mutational analysis reveals that alanine substitution of these residues impaired the anti-defense function (Fig. S7C-D).\nTogether, the data indicates that T4 and U07 TadIV-1 homologs share a conserved signal-sequestering mechanism.\n7. In figure 4C, here the WT protein completely inhibits pCaspase. How does this fit with the data in figure 2 where inhibition is only partial? Is this because a different homolog of TadIV-1 was used?\nThanks for the question.\nIn our original manuscript, metabolites prepared from T6-infected TIR-expressing cells were used to analyze the N7-cADPR binding properties of TadIV-1. The metabolites could differ in N7-cADPR concentrations, contaminants, etc, between each experiment, which might have resulted in the partial inhibition in Fig. 2. In the revised manuscript, we used commercial N7-cADPR in Fig. 2, and found that T4TadIV-1 could reduce PsCaspase activation by ~95%.\nOn the other hand, we cannot exclude the possibility that T4TadIV-1 and U07TadIV-1 may have distinct N7-cADPR affinity, given their sequence differences. Nevertheless, comparison of their N7-cADPR binding affinity is beyond the main scope of the study.\nMinor comments:\n1. Line 165 and 184 space missing\nCorrected\n2. Please add a supplemental figure showing the purified proteins, including the variants show in figure 4.\nThanks for the suggestions. An SDS-PAGE figure of purified proteins was provided (Fig. S10).\n3. The SPR data (Figure 2F) have been carried out only once. This needs to be repeated and stats provided.\nThanks for the suggestion. We repeated the SPR assay for 3 times, and the data were provided (Revised Figure 2F).\n4. Line 497 typo “incubaed”.\nCorrected\nAgain, we wish to thank the reviewer for her/his constructive comments and suggestions, which have significantly helped us to improve our manuscript.\nReviewer #2 (Remarks to the Author):\nIn the manuscript by Yu et al, the authors identified an inhibitor of type IV Thoeris named TadIV-1. They employed different methods and strategies to demonstrate that TadIV-1 functions as a sponge that sequesters the type IV Thoeris signaling molecule N7-cADPR to inhibit Caspase activation. They determined a crystal structure of apo TadIV-1, but despite numerous attempts, failed to obtain a structure of TadIV-1 bound to N7-cADPR. Structural modeling suggested a ligand binding by a flexible N-terminal loop, which was tested by mutagenesis. The authors claimed that this binding mode is distinct from other characterized sponge proteins, which form deep binding pockets between the protein subunits in oligomers (dimers, tetramers or hexamers). They also found that T6 phage can escape type IV Thoeris by the mutation in the capsid vertex protein gp224. Sponge proteins for type I and II Thoeris are quite well characterized, but information about sponges sequestering the type IV Thoeris signaling molecule is limited. Moreover, despite the numerous studies on Thoeris system, little is known about how the phage infection is recognized by ThsB (TIR) proteins. Therefore data presented in this manuscript is important and relevant to the field.\nWe thank the reviewer for recognizing the contribution of our study to the field.\nMy major concerns:\n1. Common two-helical structure of the sponge proteins. In the manuscript TadVI-1 is distinguished by a distinct binding mode, but its overall structure in comparison to other sponge proteins is not discussed. Two-helical structure is also found in 2 other anti-defense sponges: Acb2 (which was found to bind CBASSS and Thoeris signaling molecules) and Sequestin. It would be more informative in the Fig.S6 to show one subunit of the shown structures in bold color demonstrating structural differences/similarities of the sponge proteins. PDB IDs of the depicted structures should be provided in the Fig. S6 legend. To demonstrate their structural similarity and differences single subunits of Acb2, Sequestin and TadIV-1 next to each other in the same orientation should be shown (e.g. as Fig. S6B) and discussed in the manuscript. Interestingly, in Fig. S4C the N-terminal flexible loop is predicted as a beta-hairpin in a few models. Similarly, the N-terminal hairpin is present in the Acb2 proteins.\nThanks for the suggestions. We have shown one subunit of the shown structures in bold color in the revised Fig. S6 (now Fig. S8). Moreover, PDB IDs of the depicted structures have been provided in the corresponding legends. Following the reviewer’s suggestion, single subunits of Acb2, Sequestin and TadIV-1 next to each other in the same orientation have been shown in the newly added Fig. S8G and discussed in the manuscript (lines 237-246).\nIn addition, the predicted N-terminal beta-hairpin of a few TadIV-1 models is largely different from that of Acb2 proteins (Response Figure 2).\nResponse Figure 2 Comparison between TadIV-1 structural models and Acb2\n(A) A single subunit of TadIV-1 models whose N-terminal flexible loop is predicted as a β-hairpin are shown as cartoon with confidence scores marked. (B) A single subunit of Acb2 (PDB code: 8H2X).\n2. Oligomeric state of TadVI-1. Helical sponge proteins form different oligomers, from dimers, tetramers (sequestin) to hexamers (Acb2) (ref 40). Oligomeric form of TadVI-1 in solution is not known. Oligomeric structure in solution of TadIV-1 should be determined (e.g. by SEC-MALS) to demonstrate that it is a dimer in the presence and absence of N7-cADPR. A dimer of U07TadIV-1 is observed in the crystal. Can be other oligomeric structures predicted from the crystal contacts by PISA server?\nThe reviewer raised a very good point. We performed SEC-MALS analysis, which indicate that TadIV-1 exists as a dimer in solution either in the presence or absence of N7-cADPR (Fig. S4). The PISA server also predicted U07TadIV-1 as a stable dimer but no other oligomeric forms (Response Figure 3).\nResponse Figure 3 Results of PISA server using the structure of U07TadIV-1 (one chain in the asymmetric unit)\n3. Model of N7-cADPR binding. The N7-cADPR binding is predicted by boltz2 server. The main message of the manuscript is that TadVI-1 has a distinct signal binding mechanism; since the binding pocket is located not at the interface between subunits, but the binding pocket comprises the TadIV-1 N-terminal flexible loop. However, the N-terminal flexible part of TadIV-1 homologs, that inhibit type IV Thoeris, varies in length (Fig. S3). Only 3 of 8 characterized TdaIV-1 proteins contain longer variant similar to U07TadIV-1, which was characterized in detail. In the remaining 5 homologs this loop region is shorter in 9 aa residues, and it is difficult to imagine, how it could accommodate N7-cADPR in the proposed binding mode. A model of N7-cADPR binding by a TadIV-1 variant with a shorter flexible loop, like T4 TadVI-1 (Fig. S3), should be generated and compared with U07TadVI-1 predicted binding mode. Next, only residues from the flexible N-terminus (except E58) are predicted to make specific contacts with N7-cADPR (Fig. 4F, Fig. S3). Mutational analysis of these N-terminal residues of U07TadIV-1 (mutation of E58 was not performed) demonstrated that the flexible loop is important for the N7-cADPR binding. However, since in the proposed model almost no residues from the helical structure are involved in the binding, there is a possibility that the position of this flexible loop regarding the helical structure may be different from the predicted model. Other sponge proteins contain conserved positive surfaces for binding of cyclic nucleotide molecules (ref 40). The helical structure of U07TadIV-1 also contains a positive surface with the conserved residues K57, K60, H83 and a pocket next to them (in the AF3 model), and it is tempting to speculate that this surface may also be important for the N7-cADPR binding. I suggest mutating the residues of this positive interface to test their importance in N7-cADPR binding. This analysis would strengthen the proposed hypothesis. Electrostatic surface of the predicted U07TadIV-1 bound with N7-cADPR model (shown in Fig. 4E) should be shown, since N7-cADPR contains negative charge which is expected to interact with positive charge on the protein.\nWe thank the reviewer for these important suggestions. Our responses to each suggestion are listed below:\n(i) We predicted the structure of N7-cADPR-bound T4TadIV-1. This, together with our mutational studies, indicate that T4TadIV-1 binds to N7-cADPR using a similar mechanism to U07TadIV-1 (new Fig. S7). Please also refer to our response to the 6th comment of Reviewer 1.\n(II) We provided an electrostatic surface map of the predicted U07TadIV-1 (new Fig. 4I). The map reveals a positively charged region close to the N7-cADPR-binding pocket. Alanine substitution of conserved basic residues in this region, including K57, K60 and H83, also impaired the anti-defense activity. We further performed mutational analysis of K34 and E58 following the suggestion of Reviewer 3 (Please refer to the response to the 4th comment of Reviewer 3). These data uncover the roles of the conserved residues from the helical regions in N7-cADPR binding.\n4. In Fig.S2A it is shown that co-expression of TadVI-1 weakened the protection of type I Thoeris by an order of magnitude, similar to YaCBASS. It should be tested if TadIV-1 can bind type I Thoeris signaling molecule 3’cADPR.\nWe thank the reviewer for this observation. We analyzed the potential interaction of 3’-cADPR with TadVI-1 using LC, which failed to detect any direct binding (Fig. S2B).\n5. Phage escape mutant seems a bit out of topic of this manuscript. The description of gp224 and its possible recognition by Thoeris is very brief. The authors should discuss more about alternative strategies to escape antiviral defense: by acquiring anti-defense proteins of by mutating phage proteins recognized by the defense system.\nWe appreciate the reviewer’s suggestion. Indeed, phage experimental evolution can also obtain phage escapers through gain of anti-defense functions (e.g. PMID: 35924892) and other strategies. We have mentioned these possibilities and cited the references in the revised manuscript (lines 249-252).\nOn the other hand, we show that expression of a wild type version of Gp224 restored the immunity against T6 escaper. The data support Gp224 as the trigger of type IV Thoeris rather than the anti-defense function of the Gp224 mutant.\nMinor comments:\n1. Fig. 2C: the TadIV-1 concentration on X axis is shown in opposite direction (from 25 to 0). More intuitive is to use from 0 to 25.\nThanks for the suggestion. Fig. 2C has been revised.\n2. Fig. S2A: what is EV in this figure?\nThanks for the question. “EV” means the empty vector control. We have supplemented this information in the revised figure legend.\n3. Fig. 4C Lines in the legend are too thin and colors are too similar: it is difficult to follow.\nThanks for the comment. We have revised the panels (new Fig. 4C and G).\n4. Lines 225-226 “unprecedented features” sounds too dramatic.\nThanks for the suggestion. We have revised these terms throughout the manuscript.\n5. Methods: inconsistence in names (pBADHis_A / pBADHisA), units (PFU.ml-1 / PFU ml-1), etc.\nCorrected\n6. Typos: line 165 “versiondid”, line 184 “hadbound”.\nCorrected\nAgain, we wish to thank the reviewer for her/his constructive comments and suggestions, which have helped us to improve our manuscript greatly.\nReviewer #3 (Remarks to the Author):\nIn the manuscript by Chen et. al., the authors identify and characterise a novel phage-encoded sponge protein which enables escape from the type IV Thoeris phage defence system. They produce hybrid phages using hybridisation assays to produce phages comprised of genomic material from the related phages, T4 and T6. Through comparative genomics and EOP assays, the authors identify the TadIV-1 gene as responsible for the escape phenotype. PsCaspase activation assays demonstrate that this inhibition is achieved by sequestration, but not degradation, of the N7c-ADPR Thoeris type IV signalling molecule. The authors go on to solve the structure of the TadIV-1 dimer using x-ray crystallography. Though the putative N7-cADPR binding region is not resolved in the crystal structure, the authors identify regions and residues responsible for N7-cADPR binding through mutational analysis and EOP/PsCaspase assays. Further, the authors also identify a phage trigger of Thoeris type IV through directed evolution of escaper phages.\nOverall, the manuscript is a valuable contribution to the growing work on phage encoded sponge proteins which allow phage escape from a growing repertoire of defence systems which utilise cyclic nucleotide messengers. In particular, the novel binding mechanism and specificity shown by TadIV-1 is of interest. The work is technically sound, the experiments are well planned, and the manuscript is well written. I do however have a few suggestions which I believe would improve the study and ensure that all conclusions therein are thoroughly validated, along with a few minor corrections.\nWe thank the reviewer for recognizing the values of our study and for providing insightful comments.\n1. The authors identify two distinct classes of TadIV-1, long and short. While they extensively test the long version for ability to bind N7-cADPR and to inhibit Thoeris function, only one member representing the short version is tested. Could the authors please test a few additional short versions? This would add support the suggestion of the extended flexible N-terminal region being essential to N7-cADPR binding.\nThanks for the suggestion.\nWe tested 2 additional members from the short group and found that none of them could efficiently inhibit type IV Thoeris, reinforcing that the flexible N-terminal region is important for the anti-defense function (new Fig. 3C).\n2. Following on from this point, the authors also carry out a homology-based search for TadIV-1 in phage genome sequences. Are there many cases in which phages encode both short and long versions? If yes, this would support different target molecules/systems. If no, this would suggest some functional redundancy.\nThanks for the question.\nWe found that no phages encode both short and long TadIV-1s (new Table S5: List of TadIV-1 homologs). However, it’s still difficult to predict the function of the short homologs. In the revised manuscript, we do not mention the possible functional redundancy of these proteins to avoid any misleading or overstatement.\n3. The authors produce 200 binding models for N7-cADPR. In figure S4C, there does also appear to be a cluster of N7-cADPR bound between the two helices. Are there any conserved ligand poses or binding positions within this cluster? Even if not, this cluster should at least be acknowledged in text.\nThe reviewer raised a very good point. We have analyzed the 200 binding models and have found that 21 of them displayed N7-cADPR bound between the two helices. The conserved binding positions within this cluster have been described in Fig. S5D-E and acknowledged in the revised text (lines 285-288).\n4. In figure 4, additional mutational analyses should be carried out on identified binding residues from boltz2 ligand modelling. In particular, K34 and E58 account for 5 interactions in panel F. I would recommend the additional mutation combinations based on the interactions shown: K34 and E58 double mutant; S16, R18, R19, S21, S23, S24; and S16, R18, R19, S21, S23, S24, K34, E58. This would validate the ligand binding prediction and potentially show complete abrogation of N7-cADPR binding.\nThanks for the suggestion.\nWe performed the mutational analysis following the advice. Indeed, the K34/E58 double mutant exhibited decreased anti-defense activity both in vitro and in vivo (Fig. 4G-H). The combined mutations of S16, R18, R19, S21, S23, and N24 (M4) and that of S16, R18, R19, S21, S23, N24, K34, and E58 (M6) displayed a much severer effect on the sponge activity in vitro (Fig. 4G). Consistently, in phage infection assays, both the M4 and M6 mutants exhibited an almost complete loss of anti-defense activity (Fig. 4H).\n5. The authors also implicated the phage capsid vertex protein in triggering type IV Thoeris. This section should be expended on. In particular, testing for N7-cADPR levels when co-expressing the capsid vertex protein and assessing for direct binding/interactions between the capsid vertex protein and Thoeris components.\nThanks for the suggestion.\nFollowing the reviewer’s advice, we performed PsCaspase activation assay to investigate whether the T6 escaper still triggers production of N7-cADPR. The data reveal that infection by the escaper did not result in enough N7-cADPR production to activate PsCaspase (new Fig. 5B).\nWe also performed pull-down assay using purified TIR and Gp224 protein, but did not detect the direct interaction between them in vitro. The data are consistent with our observations that Gp224 alone cannot trigger the immune signaling.\nThe results have been incorporated into the revised manuscript (lines 258-265).\nMinor corrections\nIn figure 4, I find the colours in panel C difficult to interpret. I think that a bar chart of endpoint values would be clearer and would be sufficient to convey this information.\nThanks for the comment. We have revised the panels (Fig. 4C and G).\nI find the repeated use of the word “unprecedented” to be quite strong for the findings here. Perhaps “novel” or “unique” would be more appropriate.\nThanks for the suggestion. We have revised these terms throughout the manuscript.\nPlease state structure resolutions, either in text or in figure descriptions.\nThanks for the suggestion. We have stated structure resolution in the revised text.\nLine 54: Thoeris systems often cause cell growth arrest rather than direct cell death.\nIndeed, depending on the types of Thoeris systems, the hosts, or the infected phages, Thoeris may either cause cell death or cell growth arrest. We have revised the sentence to “to trigger cell dormancy or cell death and abort phage replication” (Line 52).\nLine 176: Alphafold3.\nCorrected.\nLine 410: molten, not “premelted”\nCorrected.\nPlease change degrees symbol to the correct degrees symbol throughout.\nCorrected.\nVarious spelling and grammar errors throughout methods section, please go through again before resubmission.\nThanks for the suggestion. We have read through the manuscript carefully and corrected any spelling and grammar errors.\nI hope the authors find my comments helpful and I thank them for the opportunity to read their interesting work.\nAgain, we wish to thank the reviewer for her/his constructive comments and suggestions, which have helped us to improve our manuscript greatly."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have dealt constructively with my comments, added extensive new data, and revised their interpretations.\nThe result is a significantly strengthened manuscript.\nI have no further concerns and would be happy to see this published.", "reviewer_2": "The authors have addressed all my concerns. The additional data and analyses improved the manuscript.", "reviewer_3": "Following reviewer comments, the authors have taken suggested steps to improve the manuscript. The additional mutational studies add evidence for the importance of key residues in ligand binding. Testing inhibition by additional short-tailed TadIV-1 variants added weight to the importance of flexible N-terminus region. Meanwhile, further investigation of the role of the phage capsid vertex protein in triggering Type IV Thoeris demonstrated that additional phage components are required for activation. Several additional minor changes have also been made appropriately.\nOverall, the quality of this manuscript has greatly improved, and I now recommend this article for publication."}, "author_response": "We thank the reviewer for the positive comments and for taking the time to review our manuscript.\nWe thank the reviewer for the positive comments and for taking the time to review our manuscript.\nWe thank the reviewer for the positive comments and for taking the time to review our manuscript."}]

Decision letter