CBASS

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.

News and Commentaries

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

Off

Traditional

White

Bacterial cGAS-like enzymes produce 2′,3′-cGAMP to activate an ion channel that restricts phage replication

Anonymous — Wed, 26 Jul 2023 16:44:22 +0000

Bacterial cGAS-like enzymes produce 2′,3′-cGAMP to activate an ion channel that restricts phage replication Anonymous (not verified) Wed, 07/26/2023 - 10:44

Uday Tak , Peace Holguin-Walth , Aaron Whiteley

BioRxiv (2023) PubMed PMID: 37546940; PubMed Central PMCID: PMC10402079.

Abstract

The mammalian innate immune system uses cyclic GMP–AMP synthase (cGAS) to synthesize the cyclic dinucleotide 2′,3′-cGAMP during antiviral and antitumor immune responses. 2′,3′-cGAMP is a nucleotide second messenger that initiates inflammatory signaling by binding to and activating the stimulator of interferon genes (STING) receptor. Bacteria also encode cGAS/DncV-like nucleotidyltransferases (CD-NTases) that produce nucleotide second messengers to initiate antiviral (antiphage) signaling. Bacterial CD-NTases produce a wide range of cyclic oligonucleotides but have not been documented to produce 2′,3′-cGAMP. Here we discovered bacterial CD-NTases that produce 2′,3′-cGAMP to restrict phage replication. Bacterial 2′,3′-cGAMP binds to CD-NTase associated protein 14 (Cap14), a transmembrane protein of unknown function. Using electrophysiology, we show that Cap14 is a chloride-selective ion channel that is activated by 2′,3′-cGAMP binding. Cap14 adopts a modular architecture, with an N-terminal transmembrane domain and a C-terminal nucleotide-binding SAVED domain. Domain-swapping experiments demonstrated the Cap14 transmembrane region could be substituted with a nuclease, thereby generating a biosensor that is selective for 2′,3′-cGAMP. This study reveals that 2′,3′-cGAMP signaling extends beyond metazoa to bacteria. Further, our findings suggest that transmembrane proteins of unknown function in bacterial immune pathways may broadly function as nucleotide-gated ion channels.

News and Commentaries

Read Uday's Tweetorial [Twitter]

Citation

Tak U, Walth P, Whiteley AT. Bacterial cGAS-like enzymes produce 2',3'-cGAMP to activate an ion channel that restricts phage replication.bioRxiv. 2023 Jul 24;. doi: 10.1101/2023.07.24.550367. PubMed PMID: 37546940; PubMed Central PMCID: PMC10402079.

Tak U, Walth P, ➤Whiteley AT | BioRxiv 2023

Off

Traditional

White

Molecular basis of CD-NTase nucleotide selection in CBASS anti-phage defense

Anonymous — Tue, 01 Jun 2021 06:00:00 +0000

Molecular basis of CD-NTase nucleotide selection in CBASS anti-phage defense Anonymous (not verified) Tue, 06/01/2021 - 00:00

Govande AA , Duncan-Lowey B , Eaglesham JB , Aaron Whiteley , Kranzusch PJ

Cell Reports (2021) PubMed PMID: 34077735

Abstract

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are signaling proteins that initiate antiviral immunity in animal cells and cyclic-oligonucleotide-based anti-phage signaling system (CBASS) phage defense in bacteria. Upon phage recognition, bacterial CD-NTases catalyze synthesis of cyclic-oligonucleotide signals, which activate downstream effectors and execute cell death. How CD-NTases control nucleotide selection to specifically induce defense remains poorly defined. Here, we combine structural and nucleotide-analog interference-mapping approaches to identify molecular rules controlling CD-NTase specificity. Structures of the cyclic trinucleotide synthase Enterobacter cloacae CdnD reveal coordinating nucleotide interactions and a possible role for inverted nucleobase positioning during product synthesis. We demonstrate that correct nucleotide selection in the CD-NTase donor pocket results in the formation of a thermostable-protein-nucleotide complex, and we extend our analysis to establish specific patterns governing selectivity for each of the major bacterial CD-NTase clades A-H. Our results explain CD-NTase specificity and enable predictions of nucleotide second-messenger signals within diverse antiviral systems.

Citation

Govande AA, Duncan-Lowey B, Eaglesham JB, Whiteley AT, Kranzusch PJ. Molecular basis of CD-NTase nucleotide selection in CBASS anti-phage defense. Cell Rep. 2021 Jun 1;35(9):109206. doi: 10.1016/j.celrep.2021.109206. PubMed PMID: 34077735.

Govande AA, Duncan-Lowey B, Eaglesham JB, ➤Whiteley AT, Kranzusch PJ. | Cell Reports 2021

Off

Traditional

White

CBASS

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

Abstract

News and Commentaries

Links

Citation

Off

Bacterial cGAS-like enzymes produce 2′,3′-cGAMP to activate an ion channel that restricts phage replication

Abstract

News and Commentaries

Links

Citation

Off

Molecular basis of CD-NTase nucleotide selection in CBASS anti-phage defense

Abstract

Links

Citation

Off