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Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion

Abstract

Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2–resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

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Fig. 1: DSR2 is an abortive infection protein that causes NAD+ depletion in infected cells.
Fig. 2: Genetic exchange between phages reveal regions responsible for escape from DSR2.
Fig. 3: Phage proteins that activate and inhibit DSR2.
Fig. 4: SIR2-containing defence systems deplete NAD+ in infected cells.

Data availability

Data that support the findings of this study are available within the Article and its Extended Data. Gene accessions appear in the Methods section of the paper. Plasmid maps of the constructs used for the experiments are attached as Supplementary Files. Source data are provided with this paper.

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Acknowledgements

We thank the Sorek laboratory members for comments on the manuscript and fruitful discussion. We also thank A. Brandis and T. Mehlman from the Weizmann Life Sciences Core Facilities for targeted mass spectrometry analyses, and A. Savidor from the Israel National Center for Personalized Medicine for protein mass spectrometry. R.S. was supported, in part, by the European Research Council (grant nos. ERC-CoG 681203 and ERC-AdG GA 101018520), Israel Science Foundation (grant no. ISF 296/21), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, the Deutsche Forschungsgemeinschaft (SPP 2330, grant no. 464312965), the Minerva Foundation with funding from the Federal German Ministry for Education and Research and the Knell Family Centre for Microbiology. V.S. was supported by the European Social Fund (grant no. 09.3.3-LMT-K-712-01-0126) under a grant agreement with the Research Council of Lithuania (LMTLT).

Author information

Authors and Affiliations

Authors

Contributions

J.G. and R.S. led the study and performed all analyses and experiments unless otherwise indicated. A. Lopatina performed plaque assay experiments with the SIR2/pAgo defence system. A.B. cloned and conducted plaque assays for the pVip defence system. M.Z. and V.S. provided the SIR2/pAgo defence system. S.M. and A. Leavitt cloned and conducted plaque assays for the DSR2, DSR1 and SIR2-HerA defence systems. A.M. and G.A. assisted with sequence analysis and prediction of protein domain functions and point mutations. The manuscript was written by J.G. and R.S. All authors contributed to editing the manuscript, and support the conclusions.

Corresponding author

Correspondence to Rotem Sorek.

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Competing interests

R.S. is a scientific cofounder and advisor of BiomX and Ecophage. The other authors declare no competing interests.

Peer review

Peer review information

Nature Microbiology thanks Lennart Randau, Malcolm White and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Exemplary plaque assay results showing DSR2 defence.

Plaque assay with tenfold serial dilution of phage SPR on the control B. subtilis strain (no system) or B. subtilis with DSR2.

Extended Data Fig. 2 Phage SPR does not replicate in high MOI infection of DSR2-containing cells.

Titre of phage SPR at different time points following infection of the control B. subtilis strain (no system) or B. subtilis with DSR2. Data represent plaque-forming units (PFU) per millilitre. Bar graphs represent average of three independent replicates, with individual data points overlaid.

Source data

Extended Data Fig. 3 pVip alone protects against phi3T and SPbeta but not against SPR.

Efficiency of plating (EOP) for three phages infecting the control B. subtilis strain (no system) or B. subtilis with pVip cloned from Fibrobacter sp. UWT3. Data represent plaque-forming units (PFU) per millilitre. Bar graphs represent average of three independent replicates, with individual data points overlaid. Control data are the same as those presented in Fig. 1b.

Source data

Extended Data Fig. 4 Testing of candidate genes for inhibition and activation of DSR2.

Legend of genes and operons tested included above. (a) Liquid culture growth of B. subtilis BEST7003 cells expressing either DSR2 alone, or DSR2 and a candidate gene, or control cells expressing neither gene, infected by phage SPR at 30 °C. Bacteria were infected at time 0 at an MOI of 0.04. Three independent replicates are shown for each MOI, and each curve shows an individual replicate. Each panel shows a different candidate gene. The ‘No system’ and the DSR2 control curves are the same for all 5 panels. (b, c) Transformation efficiencies of a vector containing operon 1-3 (panel B), or 4 individual genes from operon 3 (panel C) were measured for cells containing WT DSR2. Y axis represents the number of colony-forming units per millilitre obtained following transformation. Bar graphs represent average of three replicates, with individual data points overlaid.

Source data

Extended Data Fig. 5 The tail tube does not pull down DSR2 when DSAD1 is co-expressed.

Pulldown assays of the DSR2-tail tube complex, heterologously expressed in E. coli. The tail tube protein of phage SPR was C-terminally tagged with TwinStrep tag. DSR2 in this experiment was mutated (H171A) to avoid toxicity. Shown is an SDS-PAGE gel. Lane A, cells expressing tagged tail tube protein from SPR, DSR2 (H171A) and GFP. Lane B, cells expressing tagged tail tube protein from SPR, DSR2 (H171A) and DSAD1.

Source data

Extended Data Fig. 6 Multiple sequence alignment comparing the tail tube protein of SPR and SPbeta/phi3T.

The NCBI accessions for the SPR and phi3T/SPbeta tail tube proteins are WP_010328117 and WP_004399252, respectively (the phi3T tail tube protein is identical to that of SPbeta). Protein amino acid sequences were aligned by Clustal Omega30.

Extended Data Fig. 7 SIR2-containing defence systems protect against multiple phages.

a-d. Efficiency of plating (EOP) for multiple phages infecting control bacteria (no system) or bacteria expressing defence systems. Data represent plaque-forming units (PFU) per millilitre. Bar graphs represent average of three independent replicates, with individual data points overlaid. KAW1E185 is short for vB_EcoM-KAW1E185, a T4-like phage. Data appearing in this figure also appear in Fig. 4b. Data for control samples are the same in panels A and D. Asterisk marks statistically significant decreases (Student’s t-test, two-sided, P values = 0.020, 0.005, 0.036, 0.014, for phages SPR with DSR2, lambda (vir) with SIR2/pAgo, KAW1E185 with SIR2-HerA, phi29 with DSR1, respectively).

Source data

Extended Data Fig. 8 Testing for defence in mutants of SIR2/pAgo, SIR2-HerA and DSR1.

a-c. Efficiency of plating (EOP) for phages infecting the control strain (no system), bacteria containing the WT defence system, and bacteria containing mutants of the defence system. In all cases except for pAgo, point mutations were introduced in the predicted active site of the protein. For the pAgo, a bulky HSH tag, which was shown in another study to inactivate the protein31, was added to the C-terminus. Data represent plaque-forming units (PFU) per millilitre. Bar graphs represent average of three independent replicates, with individual data points overlaid. Asterisk marks statistically significant decreases (Student’s t-test, two-sided, P values = <0.001, 0.002, <0.001, for phages lambda (vir) with SIR2/pAgo, KAW1E185 with SIR2-HerA, phi29 with DSR1, respectively).

Source data

Extended Data Fig. 9 DSR1 and DSR2 alignment is limited to the SIR2 domain. SPR tail tube does not activate DSR1.

(a) Graphical representation of running BLASTP32 with a query of DSR2 and subject of DSR1. The red area spans an alignment with 23.29% identity and an E value of 9e-07. The rest of the protein is not alignable. The red bracket above indicates the predicted SIR2 domain. (b) Transformation efficiencies of a vector containing the SPR tail tube protein were measured for cells containing either DSR1 or an inactive DSR2 mutant (H171A) as control. The y axis represents the number of colony-forming units per millilitre. Bar graphs represent average of three replicates, with individual data points overlaid.

Source data

Supplementary information

Reporting Summary

Supplementary Data 1

Genomes of hybrids used in this study.

Supplementary Data 2

Plasmid file.

Supplementary Data 3

Plasmid file.

Supplementary Data

Primers used in this study.

Source data

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Garb, J., Lopatina, A., Bernheim, A. et al. Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion. Nat Microbiol 7, 1849–1856 (2022). https://doi.org/10.1038/s41564-022-01207-8

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