1.A.141.  The Cyclic Oligonucleotide-based Antiphage Signaling System Cl^- Channel Effector (CBASS-CCE) Family 

CBASS (cyclic oligonucleotide-based antiphage signaling system) immunity uses CARF-related effectors to sense 3'-5'- and 2'-5'-linked cyclic oligonucleotide signals to protect bacteria from phage infection (Lowey et al. 2020). cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Lowey et al. 2020 discovered Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is a fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. These results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling (Lowey et al. 2020). CBASS (cyclic oligonucleotide-based antiphage signaling system) provides immunity against bacteriophage. The CD-NTase protein synthesizes cyclic nucleotides in response to infection; these serve as specific second messenger signals. The signals activate a diverse range of effectors, leading to bacterial cell death and thus abortive phage infection.

Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) are a type of defence systems against bacteriophages that share ancestry with the cGAS-STING innate immune pathway in animals. CBASS systems are composed of an oligonucleotide cyclase, which generates signalling cyclic oligonucleotides in response to phage infection, and an effector that is activated by the cyclic oligonucleotides and promotes cell death (Millman et al. 2020). Cell death occurs before phage replication is completed, therefore preventing the spread of phages to nearby cells.Millman et al. 2020 analysed 38,000 bacterial and archaeal genomes and identified more than 5,000 CBASS systems, which have diverse architectures with multiple signalling molecules, effectors and ancillary genes. These authors proposed a classification system for CBASS that groups systems according to their operon organization, signalling molecules and effector functions. Four major CBASS types were identified, sharing at least six effector subtypes that promote cell death by membrane impairment, DNA degradation or other means. Evidence for extensive gain and loss of CBASS systems, as well as shuffling of effector genes between systems was obtained (Millman et al. 2020). 

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 shown to produce 2',3'-cGAMP. Tak et al. 2023 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, previously of unknown function. Using electrophysiology, Tak et al. 2023 showed 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 with 2 TMSs, 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. Thus, 2',3'-cGAMP signaling extends beyond metazoa to bacteria. These findings suggest that transmembrane proteins of unknown function in bacterial immune pathways may broadly function as nucleotide-gated ion channels.