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A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins

Aaron Whiteley — Sat, 29 Mar 2025 05:00:00 +0000

A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins Aaron Whiteley Fri, 03/28/2025 - 23:00

Ashley Sullivan , Nabhani A , Kate Schinkel , Dinh DM , Duncan ML , Ednacot EMQ , Charlotte Hoffman , Izrailevsky DS , Emily Kibby , Toni Nagy , Nguyen CM , Uday Tak , Burroughs AM , Aravind L , Aaron Whiteley , Morehouse BR

BioRxiv (2025). 2025.03.28.646047; doi: https://doi.org/10.1101/2025.03.28.646047

Abstract

Bacteria use antiphage systems to combat phages, their ubiquitous competitors, and evolve new defenses through repeated reshuffling of basic functional units into novel reformulations. A common theme is generating a nucleotide-derived second messenger in response to phage that activates an effector protein to halt virion production. Phages respond with counter-defenses that deplete these second messengers, leading to an escalating arms race with the host. Here we discover a novel antiphage system we call Panoptes that detects phage infection by surveying the cytosol for phage proteins that antagonize the nucleotide-derived second messenger pool. Panoptes is a two-gene operon, optSE. OptS is predicted to synthesize a second messenger using a minimal CRISPR polymerase (mCpol) domain, a version of the polymerase domain found in Type III CRISPR systems (Cas10) that is distantly related to GGDEF and Thg1 tRNA repair polymerase domains. OptE is predicted to be a transmembrane effector protein that binds cyclic nucleotides. optSE potently restricted phage replication but mutant phages that had loss-of-function mutations in anti-CBASS protein 2 (Acb2) escaped defense. These findings were unexpected because Acb2 is a nucleotide “sponge” that antagonizes second messenger signaling. Using genetic and biochemical assays, we found that Acb2 bound the OptS-synthesized nucleotide, 2′,3′-cyclic adenosine monophosphate (2′,3′-c-di-AMP); however, 2′,3′-c-di-AMP was synthesized constitutively by OptS and inhibited OptE. Nucleotide depletion by Acb2 released OptE toxicity thereby initiating abortive infection to halt phage replication. These data demonstrate a sophisticated immune strategy that hosts use to guard their second messenger pool and turn immune evasion against the virus.

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Read Ashley's Thread [BlueSky]

Citation

Sullivan AE, Nabhani A, Schinkel K, Dinh DM, Duncan ML, Ednacot EMQ, Hoffman CRK, Izrailevsky DS, Kibby EM, Nagy TA, Nguyen CM, Tak U, Burroughs AM, Aravind L, Whiteley AT, Morehouse BR. A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins. 2025 Mar 28; https://doi.org/10.1101/2025.03.28.646047

Sullivan AE, Nabhani A, Schinkel K, Dinh DM, Duncan ML, Ednacot EMQ, Hoffman CRK, Izrailevsky DS, Kibby EM, Nagy TA, Nguyen CM, Tak U, Burroughs AM, Aravind L, ➤Whiteley AT†, Morehouse BR† (†co-cor. author) | BioRxiv 2025

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Review: Conservation and similarity of bacterial and eukaryotic innate immunity

Anonymous — Wed, 28 Feb 2024 21:45:59 +0000

Review: Conservation and similarity of bacterial and eukaryotic innate immunity Anonymous (not verified) Wed, 02/28/2024 - 14:45

Hannah Ledvina , Aaron Whiteley

Nat Rev Microbiol (2024). PubMed PMID: 38418927; PubMed Central PMCID: PMC11389603.

Abstract

Pathogens are ubiquitous and a constant threat to their hosts, which has led to the evolution of sophisticated immune systems in bacteria, archaea and eukaryotes. Bacterial immune systems encode an astoundingly large array of antiviral (antiphage) systems, and recent investigations have identified unexpected similarities between the immune systems of bacteria and animals. In this Review, we discuss advances in our understanding of the bacterial innate immune system and highlight the components, strategies and pathogen restriction mechanisms conserved between bacteria and eukaryotes. We summarize evidence for the hypothesis that components of the human immune system originated in bacteria, where they first evolved to defend against phages. Further, we discuss shared mechanisms that pathogens use to overcome host immune pathways and unexpected similarities between bacterial immune systems and interbacterial antagonism. Understanding the shared evolutionary path of immune components across domains of life and the successful strategies that organisms have arrived at to restrict their pathogens will enable future development of therapeutics that activate the human immune system for the precise treatment of disease.

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Citation

Ledvina HE, Whiteley AT. Conservation and similarity of bacterial and eukaryotic innate immunity. Nat Rev Microbiol. 2024 Jul;22(7):420-434. doi: 10.1038/s41579-024-01017-1. Epub 2024 Feb 28. Review. PubMed PMID: 38418927; PubMed Central PMCID: PMC11389603.

Ledvina, HE, ➤Whiteley, AT | Nature Reviews Microbiology 2024

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An E1–E2 fusion protein primes antiviral immune signalling in bacteria

Anonymous — Thu, 09 Feb 2023 00:00:01 +0000

An E1–E2 fusion protein primes antiviral immune signalling in bacteria Anonymous (not verified) Wed, 02/08/2023 - 17:00

Hannah Ledvina , Ye Q , Gu Y , Ashley Sullivan , Quan Y , Lau RK , Zhou H , Corbett KD , Aaron Whiteley

Nature (2023) PubMed PMID: 36755092; PubMed Central PMCID: PMC10292035

Abstract

In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP–AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP–AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2–CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase–target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.

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Scientists discover more bacterial tools that could be reprogrammed to treat human disease [�� Today]
Read Hannah's Tweetorial [Twitter]

Citation

Ledvina HE, Ye Q, Gu Y, Sullivan AE, Quan Y, Lau RK, Zhou H, Corbett KD, Whiteley AT. An E1-E2 fusion protein primes antiviral immune signalling in bacteria. Nature. 2023 Apr;616(7956):319-325. doi: 10.1038/s41586-022-05647-4. Epub 2023 Feb 8. PubMed PMID: 36755092; PubMed Central PMCID: PMC10292035.

Ledvina HE*, Ye Q*, Gu Y, Sullivan AE, Quan Y, Lau RK, Zhou H, Corbett KD†, ➤Whiteley AT† (*equal contribution, †co-cor. author) | Nature 2023

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Spotlight Publications

A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins

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Review: Conservation and similarity of bacterial and eukaryotic innate immunity

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An E1–E2 fusion protein primes antiviral immune signalling in bacteria

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