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A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity

Nature Microbiology volume 10, pages 3162–3175 (2025) Cite this article

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Abstract

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems provide powerful adaptive immunity against phage infection. In response, phages use anti-CRISPR (Acr) proteins to evade CRISPR immunity. The few type III Acrs identified so far show conditional effectiveness in countering type III immunity or rely on unknown or poorly understood inhibitory mechanisms. Here we report the discovery of AcrIIIA2, a type III-A Acr encoded by Streptococcus thermophilus phages. Biochemical and structural analyses reveal that phage AcrIIIA2 co-opts host enolase, a highly abundant glycolysis enzyme, to form a ternary complex with the S. thermophilus type III-A (Csm) CRISPR ribonucleoprotein complex, obstructing its immune responses. The enolase-chaperoned AcrIIIA2 blocks the initial step of phage RNA binding, thereby preventing downstream type III anti-phage immune responses. Enolase participates in the anti-immune response by serving as an essential structural scaffold, stabilizing Acr–CRISPR interactions. These findings uncover a new anti-defence strategy that exploits a well-conserved host factor to block CRISPR immunity.

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Fig. 1: AcrIIIA2 was discovered from S. thermophilus phages.
The alternative text for this image may have been generated using AI.
Fig. 2: AcrIIIA2 interacts with the S. thermophilus Csm and host enolase.
The alternative text for this image may have been generated using AI.
Fig. 3: AcrIIIA2 with S. thermophilus enolase reduces target RNA binding and downstream nuclease activity.
The alternative text for this image may have been generated using AI.
Fig. 4: Cryo-EM structure of the AcrIIIA2–enolase-bound S. thermophilus Csm–crRNA complex.
The alternative text for this image may have been generated using AI.
Fig. 5: Structure and interactions of AcrIIIA2 with S. thermophilus enolase, crRNA and Csm complex.
The alternative text for this image may have been generated using AI.
Fig. 6: Model of AcrIIIA2-mediated inhibition of the S. thermophilus type III-A CRISPR complex.
The alternative text for this image may have been generated using AI.

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Data availability

The atomic coordinates of the cryo-EM structures of S. thermophilus Csm class 3:2 and class 4:3 have been deposited in Protein Data Bank under identifiers 9NO4 and 9NQ7 and in Electron Microscopy Data Bank under entries EMD-49593 and EMD-49645, respectively. The raw dataset (micrographs) and respective gain reference file have been deposited in the Electron Microscopy Public Image Archive (EMPIAR) available at https://www.ebi.ac.uk/empiar/ under accession code EMPIAR-12999. The mass spectrometry data have been deposited in the MassIVE database (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) with identifier MSV000097041 (ftp://MSV000097041@massive.ucsd.edu/). Source data are provided with this paper.

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Acknowledgements

We thank the members of the Terns and Li laboratories for the helpful discussions. Cryo-EM data were collected at the David Van Andel Advanced Cryo-Electron Microscopy Suite at the Van Andel Institute. We thank G. Zhao and X. Meng for their help with data collection. HPLC experiments were performed at Analytical Facilities at the Florida State University. We are thankful to A. Carl Whittington for assistance with HPLC experiments. This study was supported by National Institutes of Health grants R35GM118160 to M.P.T. and R35GM152081 to H.L. and National Science Foundation grant BioF:GREAT (2400220) to L.W.

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Author notes

  1. These authors contributed equally: Katie A. Johnson, Hemant N. Goswami.

Authors and Affiliations

  1. University of Georgia, Athens, GA, USA

    Katie A. Johnson, Ryan J. Catchpole, Peng Zhao, Lance Wells & Michael P. Terns

  2. Van Andel Institute, Grand Rapids, MI, USA

    Hemant N. Goswami & Hong Li

  3. Florida State University, Tallahassee, FL, USA

    Fozieh Ahmadizadeh

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  1. Katie A. Johnson
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Contributions

K.A.J. and H.N.G. contributed equally to this work. K.A.J. designed and performed experiments, analysed data, and prepared figures and the initial draft of the paper. H.N.G. performed structural analyses, contributed to experimental design and data interpretation, and prepared figures and the initial draft of the paper. R.J.C. performed bioinformatic analyses and assisted in experimental design and data interpretation. F.A. performed experiments on Csm and enolase mutational analyses. P.Z. carried out mass spectrometry experiments. L.W. supervised proteomics experiments, contributed to data interpretation and secured funding. H.L. supervised structural analyses, contributed to study design and paper writing, and secured funding. M.P.T. conceived and supervised the project, secured funding and wrote the paper with input from all authors. All authors discussed the results and commented on the paper.

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Correspondence to Hong Li or Michael P. Terns.

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Nature Microbiology thanks Ning Jia, Robert Shields and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Tables 1–4 and Figs. 1–9.

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Supplementary Video 1 (download MP4 )

Cryo-EM structure and density map of S. thermophilus Csm–crRNA complex (class 3:2) bound to AcrIIIA2 and enolase at 2.67 Å resolution. Protein subunits and RNAs are coloured as follows: AcrIIIA2, crimson; enolase, burlywood; Csm1, sky blue; Csm2, dark grey; Csm3, cornflower blue; Csm4, cadet blue; Csm5, slate blue; crRNA, green; and enolase, burlywood.

Supplementary Data 1 (download XLSX )

Mass spectrometry dataset.

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Johnson, K.A., Goswami, H.N., Catchpole, R.J. et al. A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity. Nat Microbiol 10, 3162–3175 (2025). https://doi.org/10.1038/s41564-025-02178-2

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