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Phages overcome bacterial immunity via diverse anti-defence proteins

Nature volume 625, pages 352–359 (2024)Cite this article

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Abstract

It was recently shown that bacteria use, apart from CRISPR–Cas and restriction systems, a considerable diversity of phage resistance systems1,2,3,4, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a ‘sponge’ that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.

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Fig. 1: Identification of anti-defence genes based on differential sensitivity to defence systems.
Fig. 2: The Gad1 and Gad2 proteins inhibit Gabija defence.
Fig. 3: Tad2 proteins inhibit Thoeris defence.
Fig. 4: Tad2 cancels Thoeris-mediated defence by sequestering 1″–3′ gcADPR.
Fig. 5: Had1 proteins inhibit Hachiman defence.

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Data availability

Data that support the findings of this study are available in the article and Supplementary Tables 1–15. IMG and MGV accession numbers, protein sequences and nucleotide sequences are available in Supplementary Tables 8–14. Coordinates and structure factors for cbTad1–1″–3′-gcADPR, SPO1 Tad2 apo, SPO1 Tad2–1″–3′-gcADPR, SPO1 Tad2–1″–2′-gcADPR and Had1 have been deposited in the Protein Data Bank under the accession codes 8SMD, 8SME, 8SMF, 8SMG and 8TTO, respectively. The genome sequences of the phages SPO1L1–SPO1L5 and SPβL1–SPβL8 have been deposited with GenBank under accession codes OQ921336–OQ921348, respectively. Source data are provided with this paper.

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Acknowledgements

We thank the members of the laboratory of R.S. for comments on the manuscript and discussion; Y. Peleg and S. Albeck for assistance with protein expression; Y. Fridmann-Sirkis for help with surface plasmon resonance analysis; and H. Keren-Shaul and D. Pilzer for help with PacBio sequencing. R.S. was supported, in part, by the European Research Council (grant no. ERC-AdG GA 101018520), the Israel Science Foundation (MAPATS grant 2720/22), the Deutsche Forschungsgemeinschaft (SPP 2330, Grant 464312965), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, and the Knell Family Center for Microbiology. E.Y. is supported by the Clore Scholars Program, and, in part, by the Israeli Council for Higher Education via the Weizmann Data Science Research Center. P.J.K. was supported, in part, by the Pew Biomedical Scholars programme and The Mathers Foundation. S.J.H. is supported through a Cancer Research Institute Irvington Postdoctoral Fellowship (number CRI3996). X-ray data were collected at the Northeastern Collaborative Access Team beamlines 24-ID-C and 24-ID-E (P30 GM124165), and used a Pilatus detector (S10RR029205), an Eiger detector (S10OD021527) and the Argonne National Laboratory Advanced Photon Source (DE-AC02-06CH11357), and at beamline 8.2.1 of the Advanced Light Source, a US Department of Energy Office of Science User Facility under contract number DE-AC02-05CH11231 and supported in part by the Howard Hughes Medical Institute, the ALS-ENABLE programme and the NIGMS grant P30 GM124169-01.

Author information

Author notes

  1. These authors contributed equally: Erez Yirmiya, Azita Leavitt

Authors and Affiliations

  1. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Erez Yirmiya, Azita Leavitt, Carmel Avraham, Ilya Osterman, Jeremy Garb, Gil Amitai & Rotem Sorek

  2. Department of Microbiology, Harvard Medical School, Boston, MA, USA

    Allen Lu, Adelyn E. Ragucci, Sadie P. Antine, Sarah E. Mooney, Samuel J. Hobbs & Philip J. Kranzusch

  3. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA

    Allen Lu, Adelyn E. Ragucci, Sadie P. Antine, Sarah E. Mooney, Samuel J. Hobbs & Philip J. Kranzusch

  4. Parker Institute for Cancer Immunotherapy at Dana-Farber Cancer Institute, Boston, MA, USA

    Philip J. Kranzusch

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  1. Erez Yirmiya
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Contributions

The study was conceptualized and designed by E.Y., A. Leavitt and R.S. E.Y. built and executed the computational pipeline and analysed the data. A. Leavitt isolated the phages and conducted all of the in vivo experiments unless stated otherwise. A. Lu and P.J.K. carried out the structural analysis of Tad1 and Tad2. A.E.R. and P.J.K. determined and analysed the structure of Had1. C.A. and G.A. carried out the biochemical experiments with cell lysates and led the mechanistic characterization of the Tad2 activity. I.O. designed and conducted all of the phage knock-in experiments and the knockout of gad2 from the phage SPβL7. J.G. designed and generated the knockdown clones. DNA cleavage experiments were carried out by S.P.A., S.E.M. and P.J.K. S.J.H. helped with the structural analysis, characterization of the Tad2 activity, and analysis of Had1 oligomerization. The study was supervised by G.A. and R.S. The manuscript was written by E.Y. and R.S. All authors contributed to editing the manuscript and support the conclusions.

Corresponding authors

Correspondence to Gil Amitai or Rotem Sorek.

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Competing interests

R.S. is a scientific co-founder of and adviser for BiomX and Ecophage. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Genome comparisons of phages from the SPbeta and SBSphiJ groups.

Genome comparison of (a) eleven phages from the SPbeta group and (b) eight phages from the SBSphiJ group. Amino acid sequence similarity is marked by grey shading. Genome similarity was visualized using clinker56.

Extended Data Fig. 2 Phages from the same family are differentially sensitive to bacterial defense systems.

Results of phage infection experiments with (a) eleven phages of the SPbeta group, (b) six phages of the SPO1 group, and (c) eight phages of the SBSphiJ group. Data represent plaque-forming units per ml (PFU/ml) of phages infecting control cells (“no system”), and cells expressing the respective defense systems. Shown is the average of three technical replicates, with individual data points overlaid. The Thoeris and Hachiman data presented here are the same as those presented in Figs. 3b and 5b, respectively.

Source Data

Extended Data Fig. 3 Gad1 proteins inhibit Gabija mediated defense.

(a) Multiple sequence alignment of the original Gad1 from phage phi3T and five Gad1 homologs that were chosen for experimental verification. Conserved residues are in purple. (b) Results of phage infection experiments with eleven phages of the SPbeta group. Data represent plaque-forming units per ml (PFU/ml) of phages infecting control cells (“no system”), cells expressing the Gabija system (“Gabija”), and cells co-expressing the Gabija system and a Gad1 homolog. Shown is the average of three technical replicates, with individual data points overlaid. The SPbeta data presented here are the same as those presented in Fig. 2d.

Source Data

Extended Data Fig. 4 Gad2 inhibits Gabija mediated defense.

(a) Phylogeny and distribution of Gad2 homologs. Homologs that were tested experimentally are indicated on the tree by cyan diamonds. (b) An Alphafold2 model for the structure of Gad2 from phage SPbetaL7. (c) Mutations in the predicted nucleotidyltransferase active site in Gad2 result in loss of anti-defense activity. Data represent plaque-forming units per ml (PFU/ml) of phage SPbeta infecting cells co-expressing the Gabija system and WT or mutated Gad2 from Brevibacillus laterosporus, as well control cells expressing neither Gabija nor Gad2 (“Control”) and cells expressing the Gabija system without Gad2 (“No Gad2”). Shown is the average of three technical replicates, with individual data points overlaid. (d) SDS-PAGE analysis of Ni-NTA co-purified GajAB with Shewanella phage 1/4 Gad1 and Brevibacillus laterosporus Gad2 demonstrates that Gad2 does not stably interact with the GajAB complex. Asterisk indicates minor contamination with the E. coli protein ArnA. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. (e) SDS-PAGE analysis of purified Brevibacillus laterosporus Gad2. Asterisk indicates contamination with the E. coli protein ArnA. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. (f) Biochemical reconstitution of GajAB DNA degradation demonstrates that Gad2 does not directly inhibit GajAB cleavage of a 56-bp target DNA. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 5 Tad2 proteins inhibit Thoeris mediated defense.

(a) Multiple sequence alignment of the original Tad2 from phage SPO1, and 5 Tad2 homologs that were chosen for experimental verification. Conserved residues are in purple. Black arrows indicate residues that are involved in 1″–3′ gcADPR binding. (b) Results of phage infection experiments with six phages of the SPO1 group. Data represent plaque-forming units per ml (PFU/ml) of phages infecting control cells (“no system”), cells expressing the Thoeris system (“Thoeris”), and cells co-expressing the Thoeris system and a Tad2 homolog. Shown is the average of three technical replicates, with individual data points overlaid.

Source Data

Extended Data Fig. 6 Tad2 binds 1″–3′ gcADPR.

(a) Incubation of Tad2 with 1″–3′ gcADPR in vitro does not yield observable degradation products. Representative HPLC traces of 1″–3′ gcADPR incubated with buffer, Tad2, or with the enzyme Cap-Clip known to cleave diphosphate linkages as a positive control. (b) Size-exclusion chromatography of 1″–3′ gcADPR-bound or apo state Tad2. 1″–3′ gcADPR-bound Tad2 shows a substantial shift compared to Tad2 in the apo state. (c) Surface plasmon resonance binding sensorgrams for Tad2 at five concentrations of 1″–3′ gcADPR. The black lines are the global fits using the instrument’s evaluation software. ka = 3.42E + 05 ± 5.2E + 02 (1/Ms), kd = 0.00798 ± 1E-05 (1/s). (d) Surface plasmon resonance binding sensorgrams for Tad2 at multiple concentrations of cADPR.

Source Data

Extended Data Fig. 7 Size-exclusion chromatography of Tad2 and various standards.

Observed peak demonstrates that Tad2 forms a homomultimer.

Source Data

Extended Data Fig. 8 Comparison of Tad2 and Tad1 in the apo and ligand-bound states.

(a) Overview of the crystal structure of SPO1 Tad2 in the apo state in front and top view. (b,c) Overview and detailed binding pocket views of adenine interactions (left) and ribose/phosphate interactions (right) of the crystal structures of SPO1 Tad2 in complex with 1″–3′ gcADPR (b) or 1″–2′ gcADPR (c). (d) Overview of the crystal structure (PDB: 7UAV) of cbTad1 in the apo state in front view and top view. (e,f) Overview and detailed binding pocket views of adenine interactions (left) and ribose/phosphate interactions (right) of the crystal structures of cbTad1 in complex with 1″–3′ gcADPR (e) or 1″–2′ gcADPR (f, PDB: 7UAW).

Extended Data Fig. 9 Had1 proteins inhibit Hachiman-mediated defense.

(a) Results of phage infection experiments with eight phages of the SBSphiJ group. Data represent plaque-forming units per ml (PFU/ml) of phages infecting control cells (“no system”), cells expressing the Hachiman system (“Hachiman”), and cells co-expressing the Hachiman system and a Had1 homolog. Shown is the average of three technical replicates, with individual data points overlaid. (b) Structure-guided sequence alignment of Had1 homologs colored by BLOSUM62 score. (c) SDS-PAGE and (d) SEC-MALS analysis of purified Had1. Full-length Had1 elutes as a single species that is consistent with a homodimeric complex (predicted homodimer 12.5 kDa, observed 12.6 kDa). Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Table 1 Summary of crystallography data collection, phasing, and refinement statistics
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Supplementary information

Supplementary Fig. 1

Uncropped gel images.

Reporting Summary

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Supplementary Tables 1

Supplementary Tables 1–6.

Supplementary Tables 2

Supplementary Tables 7–14.

Supplementary Table 15

Supplementary Table 15.

Source data

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Yirmiya, E., Leavitt, A., Lu, A. et al. Phages overcome bacterial immunity via diverse anti-defence proteins. Nature 625, 352–359 (2024). https://doi.org/10.1038/s41586-023-06869-w

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