Nabhani, A. | Search bioRxiv

bioRxiv2026

Minimal diadenylate cyclases have been co-opted to detect phage immune evasion

Many bacterial immune defenses transmit recognition of phage infection via the generation of diverse cyclic nucleotide second messengers. Phage have evolved to subvert this kind of immunity by sequestering, degrading, or inhibiting synthesis of these signaling molecules. Consequently, bacteria have evolved counter-mechanisms to detect disruption of cyclic nucleotide signaling and induce another layer of immune protection. Here we detail our discovery of the PanDA defense system (Panoptes-like DisA), an antiphage defense which detects sequestration of 3'3'-c-di-AMP by phage sponge proteins. PanDA consists of two proteins, PanS and PanE, which are both necessary for defense. PanS contains a minimal diadenylate cyclase (DAC) domain that constitutively generates the cyclic dinucleotide 3'3'-c-di-AMP which binds to and represses a toxic effector, the 2TM-{beta} family protein PanE. When a cell is infected by a phage encoding the sponge protein Acb4 (anti-CBASS protein 4), PanE is activated and induces membrane permeability. This work represents the first confirmed use of 3'3'-c-di-AMP as an immune second messenger in bacteria, facilitated by the exaptation of a DAC domain which has thus far only been best understood for its non-immune signaling roles.

microbiology↗

bioRxiv2025

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

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.

microbiology↗