Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages

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Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector’s toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr’s pivotal role in immunity.
OriginalsprogEngelsk
TidsskriftMolecular Cell
Vol/bind84
Udgave nummer11
Sider (fra-til)2185-2202.e12
Antal sider31
ISSN1097-2765
DOI
StatusUdgivet - 2024

Bibliografisk note

Funding Information:
We thank the Danish cryo-EM National Facility in CFIM (University of Copenhagen) and Nicholas Sofos for cryo-EM data collection support. We thank Raissa Novais, Mansa Nair-Kihara, and Thomas Moritz (Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen) for their help in preliminary metabolomic experiments. We acknowledge Feng Zhang (Broad Institute and MIT, Cambridge) for kindly providing pLG001 and pLG007 plasmids (Addgene plasmids #157879 and #157885). We thank Lars Hansen and Nikoline S. Olsen (Department of Plant and Environmental Sciences, PLEN, University of Copenhagen), Jakob Bobonis (Typas group, EMBL), and Claudia Igler (ETH, Zurich) for kindly providing the coliphages used here. We thank S\u00F8ren J. S\u00F8rensen for generously granting us access to the laboratory space and technical equipment at the Section of Microbiology (University of Copenhagen). This work was supported by Lundbeck Foundation grants R347-2020-2346 and R380-2021-1448 awarded to R.P.-R. and A.C. respectively. G.M. and M.L.N. are part of the Novo Nordisk Foundation Center for Protein Research (NNF-CPR), which is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001). M.L.N. is also supported by The Danish Council of Independent Research (0135-00096B, 2034-00311B, and 2032-00311B), The Danish Cancer Society (R146-A9159-16-S2), and the European Union's Horizon 2020 research and innovation program under grant agreement EPIC-XS-823839. This work was also supported by the NNF0024386, NNF17SA0030214, Distinguished Investigator NNF18OC0055061, Distinguished Innovator NNF23OC0082600, and ERC_AdG_101096548 INTETOOLS grants to G.M. who is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. Conceptualization, A.C. S.C.-W. M.R.M. R.P.-R. and G.M.; methodology, A.C. S.C.-W. M.R.M. B.L.-M. and I.A.H.; software, M.R.M.; investigation, A.C. S.C.-W. M.R.M. B.L.-M. I.A.H. R.Z. T.P. A.F. and S.H.-C.L.; resources, G.M. and M.L.N.; data curation, A.C. M.R.M. B.L.-M. and I.A.H.; writing \u2013 original draft, A.C. R.P.-R. and G.M.; writing \u2013 review & editing, A.C. R.P.-R. and G.M.; visualization, A.C. S.C.-W. M.R.M. B.L.-M. and I.A.H.; supervision, A.C. R.P.-R. M.L.N. and G.M.; project administration, A.C. R.P.-R. M.L.N. and G.M.; funding acquisition, A.C. R.P.-R. M.L.N. and G.M. G.M. is a stockholder and member of the SAB of Ensoma, and M.R.M. is listed as co-inventor of patent application WO2023141602A2, related to the use of engineered retrons for genome editing.

Funding Information:
We thank the Danish cryoEM National Facility in CFIM (University of Copenhagen) and Nicholas Sofos for cryo-EM data collection support. We thank Raissa Novais, Mansa Nair-Kihara, and Thomas Moritz (Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen) for their help in preliminary metabolomic experiments. We acknowledge Feng Zhang (Broad Institute and MIT, Cambridge) for kindly providing pLG001 and pLG007 plasmids (Addgene plasmids #157879 and #157885). We thank Lars Hansen and Nikoline S. Olsen (Department of Plant and Environmental Sciences, PLEN, University of Copenhagen), Jakob Bobonis (Typas group, EMBL), and Claudia Igler (ETH, Zurich) for kindly providing the coliphages used here. We thank S\u00F8ren J. S\u00F8rensen for generously granting us access to the laboratory space and technical equipment at the Section of Microbiology (University of Copenhagen). This work was supported by Lundbeck Foundation grants R347-2020-2346 and R380-2021-1448 awarded to R.P.-R. and A.C., respectively. G.M. and M.L.N. are part of the Novo Nordisk Foundation Center for Protein Research (NNF-CPR), which is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001 ). M.L.N. is also supported by The Danish Council of Independent Research ( 0135-00096B , 2034-00311B , and 2032-00311B ), The Danish Cancer Society ( R146-A9159-16-S2 ), and the European Union\u2019s Horizon 2020 research and innovation program under grant agreement EPIC-XS-823839 . This work was also supported by the NNF0024386 , NNF17SA0030214 , Distinguished Investigator NNF18OC0055061 , Distinguished Innovator NNF23OC0082600, and ERC_AdG_101096548 INTETOOLS grants to G.M. who is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen.

Publisher Copyright:
© 2024 Elsevier Inc.

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