Abstract
Bacteria defend against phage infection through a variety of antiphage defence systems1. Many defence systems were recently shown to deplete cellular nicotinamide adenine dinucleotide (NAD+) in response to infection, by cleaving NAD+ into ADP-ribose (ADPR) and nicotinamide2,3,4,5,6,7. It was demonstrated that NAD+ depletion during infection deprives the phage of this essential molecule and impedes phage replication. Here we show that a substantial fraction of phages possess enzymatic pathways allowing reconstitution of NAD+ from its degradation products in infected cells. We describe NAD+ reconstitution pathway 1 (NARP1), a two-step pathway in which one enzyme phosphorylates ADPR to generate ADPR pyrophosphate (ADPR-PP), and the second enzyme conjugates ADPR-PP and nicotinamide to generate NAD+. Phages encoding NARP1 can overcome a diverse set of defence systems, including Thoeris, DSR1, DSR2, SIR2–HerA and SEFIR, all of which deplete NAD+ as part of their defensive mechanism. Phylogenetic analyses show that NARP1 is primarily encoded on phage genomes, suggesting a phage-specific function in countering bacterial defences. A second pathway, NARP2, allows phages to overcome bacterial defences by building NAD+ using metabolites different from ADPR-PP. Our findings reveal a unique immune evasion strategy in which viruses rebuild molecules depleted by defence systems, thus overcoming host immunity.
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Data availability
Data that support the findings of this study are available within the Article and Supplementary Tables 1–4. IMG and INPHARED accession numbers, protein sequences and nucleotide sequences are available in Supplementary Tables 1 and 2. Source data are provided with this paper.
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Acknowledgements
We thank members of the laboratory of R.S. for comments on earlier versions of this manuscript. R.S. was supported, in part, by the European Research Council (grant number ERC-AdG GA 101018520), the Israel Science Foundation (MAPATS grant 2720/22), the Deutsche Forschungsgemeinschaft (SPP 2330, grant 464312965), a research grant from the Estate of Marjorie Plesset, the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, the Dr. Barry Sherman Institute for Medicinal Chemistry, M. de Botton, the Andre Deloro Prize and the Knell Family Center for Microbiology. I.O. was supported by the Ministry of Absorption New Immigrant programme. E.Y. is supported by the Clore Scholars Program, and, in part, by the Israeli Council for Higher Education through the Weizmann Data Science Research Center.
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The study was conceptualized and designed by I.O. and R.S. I.O. conducted all phage knockout and knock-in experiments, tested the anti-defence effect of NARP1, purified Adps and Namat proteins and carried out all in vitro experiments. H.S. cloned and measured the anti-defence effect of NARP2. F.R. carried out a phylogenetic analysis of Adps and Namat. E.L. collected and processed samples for LC–MS. M.I. and S.M. carried out all LC–MS experiments. E.Y. identified phages with NARP1 in laboratory phage collection. A.M. collected sequences of Prs and Nampt genes in bacterial and phage genomes. The manuscript was written by I.O. and R.S. All authors contributed to editing the manuscript and support the conclusions.
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R.S. is a scientific cofounder and adviser of BiomX and Ecophage. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 BASEL-collection phages encoding NAD reconstitution pathways.
a. BASEL-collection phages that encode the NARP1 pathway. b. BASEL-collection phages that encode a two-gene operon predicted to comprise NadR and a transporter for nicotinamide riboside. This operon is hypothesized to comprise a phage NAD+ reconstitution pathway that was not examined in the current study.
Extended Data Fig. 2 Mass spectrometry analysis of metabolites detected in lysates from infected cells.
a. A unique molecule with an m/z value of 622.034 appears in SIR2-HerA cells infected by Bas63. Cells were infected at MOI = 10. Bars represent the mean area under the curve (AUC) of three experiments, with individual data points overlaid. b. Extracted mass chromatograms of ions with an m/z value of 622.034 (positive ionization mode) and 620.021 (negative ionization mode) and retention time of 10.2 min. c. MS data in negative ionization mode for the same molecule presented in Fig. 2b. d. MS/MS fragmentation spectra of the molecule with the m/z value 620.021 (negative ionization mode) and 622.034 (positive ionization mode). The hypothesized structure of the molecule and MS/MS fragments are presented.
Extended Data Fig. 3 Characterization of the Adps and Namat enzymes.
a. An SDS-PAGE gel showing the purified Adps (predicted Mw of 32.1 kDa including the 6xhis tag) and Namat (56.6 kDa including the 6xhis tag). For gel source data, see Supplementary Fig. 1. b. A plot of the velocity (µmol* min−1*mg−1) of AMP production by Adps in various concentrations of ADPR in the presence of 100 µM ATP. c. A plot of the velocity (µmol*min−1*mg−1) of AMP production by Adps in various concentrations of ATP in the presence of 100 µM ADPR. In both panels b and c, the appearance of AMP was measured by the AMP-Glo assay (Promega) d. A plot of the velocity (µmol*min−1*mg−1) of NAD production by Namat in various concentrations of nicotinamide in the presence of 100 µM ADPR-PP. e. A plot of the velocity (µmol*min−1*mg−1) of NAD production by Namat in various concentrations of ADPR-PP in the presence of 100 µM nicotinamide. Steady-state kinetic parameters were evaluated by fitting the Michaelis-Menten or Hill equations to data generated by 3 independent measurements using the Enzyme_Kinetics_Calculator47. Individual data points are overlaid on the curves.
Extended Data Fig. 4 Enzymatic treatment of NARP1 products.
a. Schematic of the reactions and mass-chromatograms of ADPR-PP and ADPR-cP following incubation with the enzyme NudC. Representative chromatograms of three replicates are presented. b. Schematic of the reactions and mass-chromatograms of ADPR-PP and ADPR-cP following incubation with the enzyme Apyrase. The peak with m/z 622.034 and retention time 11.0 is hypothesized to correspond to fragmentation of ADPR-PP by ionization in mass spectrometer. Representative chromatograms of three replicates are presented.
Supplementary information
Supplementary Fig. 1
Uncropped protein gel of the results of the Adps and Namat purification.
Supplementary Table 1
List of Adps homologues.
Supplementary Table 2
List of Namat homologues.
Supplementary Table 3
List of strains, plasmids and phages used in this study.
Supplementary Table 4
List of primers used in this study.
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Osterman, I., Samra, H., Rousset, F. et al. Phages reconstitute NAD+ to counter bacterial immunity. Nature (2024). https://doi.org/10.1038/s41586-024-07986-w
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DOI: https://doi.org/10.1038/s41586-024-07986-w
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