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Expression level of anti-phage defence systems controls a trade-off between protection range and autoimmunity

Nature Microbiology volume 10, pages 1954–1962 (2025)Cite this article

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Abstract

The evolutionary arms race between bacteria and phages has given rise to elaborate anti-phage defence mechanisms. Although many of these systems have been characterized at the molecular level, the general principles and constraints at play are underexplored. It is broadly recognized that in addition to the protection they provide, these systems also bear a substantial cost. Here we identify an expression-dependent trade-off between the protection range of defence systems and the fitness burden they impose. We first focus on the SpbK system of Bacillus subtilis and then generalize to other systems across a range of bacteria. We show that increasing expression of defence systems enhances their protection range, and provide evidence that this is achieved by overwhelming phage strategies for circumventing bacterial defence. However, for most systems tested, increased expression also leads to self-inflicted toxicity. This trade-off between protection and autoimmunity may shape the evolution of regulatory strategies and favour the coexistence of multiple systems within a single genome.

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Fig. 1: Overexpression of the spbK defence system extends its protection range.
Fig. 2: Extended protection range upon overexpression occurs in multiple defence systems and depends on overcoming different counter-defence strategies.
Fig. 3: Defence overexpression leads to autoimmunity in multiple defence systems.
Fig. 4: Illustration of the expression-controlled trade-off between the benefit of protection range and the cost of autoimmunity.

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Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

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Code used to generate all figures is provided in the supplementary files.

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Acknowledgements

We thank S. Pollak and J. Garb for fruitful discussions, and members of the Eldar lab for comments on the paper. We also thank T. Mahata, U. Qimron and D. Salomon for providing GAPS4 strains and for fruitful discussions. The Eldar lab is funded by European Research Council grant 101118890 and by Israel Science Foundation grant 2228/21. N.A. was supported by the Israeli Academy of Science’s Adams Fellowship.

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Author notes

  1. These authors contributed equally: Nitzan Aframian, Shira Omer Bendori.

Authors and Affiliations

  1. Shmunis School of Biomedicine and Cancer Research, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

    Nitzan Aframian, Shira Omer Bendori, Tal Hen, Polina Guler & Avigdor Eldar

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  1. Nitzan Aframian
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  2. Shira Omer Bendori
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Contributions

A.E. and N.A. designed the study. All experiments were performed by S.O.B unless indicated otherwise. S.O.B., T.H. and N.A. performed phage infection experiments. S.O.B. and P.G. performed RT–qPCR experiments. A.E. and N.A. wrote the paper. A.E. supervised the study.

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Correspondence to Avigdor Eldar.

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Nature Microbiology thanks Edze Westra and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Native and induced spbK relative expression levels.

(a) Relative transcript levels of spbK under different conditions, measured by RT-qPCR (methods). spbKICE is a strain harboring the full ICEBs1 element, and spbKwt a strain harboring spbK alone under its native promoter. Shown are means and error bars represent s.e.m. Points represents n = 3 biological repeats. (b) YFP expression under an IPTG-inducible promoter at different concentrations of IPTG. Shown are geometric means and s.e.m of the log. Individual points represent n = 3 biological repeats.

Extended Data Fig. 2 PFU/ml of different phages 2 hrs post infection of strains harboring spbK at different expression levels.

Shown are geometric means and s.e.m of the log. Individual points represent n = 3 biological repeats. * marks p < 0.05, ** marks p < 0.005, and *** marks p < 0.0005 (two-sided t-test).

Extended Data Fig. 3 Growth curves of bacterial strains expressing spbK at different expression levels, infected by different phages at MOI = 0.1.

Mean OD of n = 3 biological repeats and s.e.m (lighter shade) are shown.

Extended Data Fig. 4 Growth curves of cells coexpressing the natively expressed spbK gene and xylose-induced portal proteins of either phage SPO1 (gp3.3; left-hand side) or ϕ105 (gp04; right-hand side).

Mean OD of n = 3 biological repeats and s.e.m (lighter shade) are shown.

Extended Data Fig. 5 Growth curves of bacterial strains expressing Gabija at different expression levels, infected by different phages at MOI = 0.1.

Mean OD of n = 3 biological repeats and s.e.m (lighter shade) are shown.

Extended Data Fig. 6 Growth curves of bacterial strains expressing Lamassu at different expression levels, infected by different phages at MOI = 0.1.

Mean OD of n = 3 biological repeats and s.e.m (lighter shade) are shown.

Extended Data Fig. 7 Growth curves of bacterial strains expressing Septu at different expression levels, infected by different phages at MOI = 0.1.

Mean OD of n = 3 biological repeats and s.e.m (lighter shade) are shown.

Extended Data Fig. 8 PFU/ml of different phages 2 hrs post infection of strains harboring different system at different expression levels.

(a) Natively-expressed Lamassu shows weak but statistically significant effect against phages ϕ3T, ϕ3T-vir, and SPR. All strains were supplemented with 2% xylose. (b) Septu protects against SPO1 when overexpressed but not under native expression. Notice that protection against ϕ3T under native expression is only significant when applying a one-sided rather than two-sided t-test. All strains were supplemented with 50 μM IPTG. Shown are geometric means and s.e.m of the log. Individual points represent n = 3 biological repeats. * marks p < 0.05, and ** marks p < 0.005 (two-sided t-test).

Extended Data Fig. 9 Growth curves of E. coli bacterial strains expressing GAPS4 at different expression levels, infected by different phages at MOI = 0.1.

Mean OD of n = 3 biological repeats and s.e.m (lighter shade) are shown.

Extended Data Fig. 10 PFU/ml of EOP assays of different phages infecting strains expressing dsr2 at different levels.

Shown are geometric means and s.e.m of the log for phage SPR (a), an evading SPR-SPβ hybrid (b), a neutralizing SPR-SPβ hybrid (c) and phage SPβ (d). Individual points represent n = 3 biological repeats.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4.

Reporting Summary

Supplementary Tables 1–3

1. Strain list. 2. Plasmid list. 3. Primer list.

Supplementary Data

Directory with data and code used to generate all figures, including all data files and code, as well as plate reader data.

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Aframian, N., Omer Bendori, S., Hen, T. et al. Expression level of anti-phage defence systems controls a trade-off between protection range and autoimmunity. Nat Microbiol 10, 1954–1962 (2025). https://doi.org/10.1038/s41564-025-02063-y

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