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 Preprint, July 24 2023, https://doi.org/10.1101/2023.07.24.550367

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

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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 , ➤Whiteley AT , Kranzusch PJ

Cell Rep. 2021 Jun 1;35(9):109206. doi: 10.1016/j.celrep.2021.109206.

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.

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