DnaJ mediates phage sensing by the bacterial NLR-related protein bNACHT25

Aaron Whiteley — Fri, 30 May 2025 15:00:00 +0000

DnaJ mediates phage sensing by the bacterial NLR-related protein bNACHT25 Aaron Whiteley Fri, 05/30/2025 - 09:00

Amy Conte , Madison Ruchel , Samantha Ridgeway , Emily Kibby , Toni Nagy , Aaron Whiteley

PLoS Biology (2025). PubMed PMID: 40446071; PubMed Central PMCID:

Abstract

Bacteria encode a wide range of antiphage systems and a subset of these proteins are homologous to components of the human innate immune system. Mammalian nucleotide-binding and leucine-rich repeat containing proteins (NLRs) and bacterial NLR-related proteins use a central NACHT domain to link detection of infection with initiation of an antimicrobial response. Bacterial NACHT proteins provide defense against both DNA and RNA phages. Here we investigate the mechanism of phage detection by the bacterial NLR-related protein bNACHT25 in E. coli. bNACHT25 was specifically activated by Emesvirus ssRNA phages and analysis of MS2 phage escaper mutants that evaded detection revealed a critical role for Coat Protein (CP). A genetic assay showed CP was sufficient to activate bNACHT25 but the two proteins did not directly interact. Instead, we found bNACHT25 requires the host chaperone DnaJ to detect CP and protect against phage. Our data support a model in which bNACHT25 detects a wide range of phages using an indirect mechanism that may involve guarding a host cell process rather than binding a specific phage-derived molecule.

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Conte AN, Ruchel ME, Ridgeway SM, Kibby EM, Nagy TA, Whiteley AT. DnaJ mediates phage sensing by the bacterial NLR-related protein bNACHT25. PLoS Biol. 2025 May 30;23(5):e3003203. doi: 10.1371/journal.pbio.3003203. [Epub ahead of print] PubMed PMID: 40446071.

Conte AN, Ruchel ME, Ridgeway SM, Kibby EM, Nagy TA, ➤Whiteley, AT | PLoS Biology 2025

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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.

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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

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DnaJ mediates phage sensing by the bacterial NLR-related protein bNACHT25

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

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