Excited to share that my work from the @aaronwhiteley.bsky.social lab is now on bioRxiv! With @benmorehouse.bsky.social, we discovered that the Panoptes defense system—named after the all-seeing watchman of Hera—uses decoy nucleotides to detect phage anti-defense proteins.

Nucleotide-based signaling is a hallmark of many antiphage systems (CBASS, Pycsar, Thoeris, etc.). Here we investigated Panoptes, a two-gene operon (optSE) that we predicted would similarly use nucleotide second messengers to carry out an immune response.

We first explored the antiphage activity of Panoptes and found that it robustly and specifically defended against Teven phages from the Straboviridae family.

To better understand the mechanism of this system, we determined a crystal structure of OptS and found that it resembled the catalytic regions of both Cas10 and GGDEF enzymes and formed a stable tetrameric complex.

We tested the activity of OptS by incubating it with a range of NTPs and found that it used ATP as a substrate to specifically produce 2′,3′-c-di-AMP, an isomer of c-di-AMP. We used a series of phosphodiesterases to show that the linkage included a 3′–5′ and 2′–5′ bond.

Intriguingly, the escaper phages we identified encoded loss-of-function mutations exclusively in the anti-CBASS 2 (acb2) gene. Consistent with this, we found that acb2 was necessary and sufficient to activate Panoptes defense.

Acb2 was recently discovered by the Bondy-Denomy and Chen Labs to be an anti-defense protein that acts as a nucleotide “sponge,” sequestering CBASS-derived cyclic oligonucleotide signaling molecules.

These findings left us puzzled: How does the Acb2 nucleotide sponge protein activate the Panoptes system? Based on its ability to bind diverse nucleotide products, we proposed that Acb2 also binds the product of OptS (2′,3′-c-di-AMP), which is exactly what we found.

These data led us to hypothesize that OptS-synthesized nucleotides were actually inhibiting, rather than activating, the OptE effector. Upon phage infection, however, Acb2 sequesters the OptS product away from OptE, unleashing its growth inhibition effect.

Consistent with this, we found that OptE robustly inhibited bacterial growth unless an OptS-derived nucleotide was present and that OptS constitutively synthesizes 2′,3′-c-di-AMP in vivo in the absence of phage infection.

Finally, we wanted to explore the relationship between CBASS and Panoptes, given the importance of Acb2 in both nucleotide-based signaling systems. We found that 53% of all Panoptes systems co-occur with CBASS, suggesting that Panoptes systems are predominantly guardians of CBASS in bacteria.

In total, we uncovered a novel mechanism of immune evasion sensing in bacteria. The Panoptes system is part of a “layered” immune system that flips the liability of a nucleotide-derived second messenger in immune signaling against the phage, adding an interesting dimension to phage-bacteria warfare.

Huge shout-out to all of the authors from the @aaronwhiteley.bsky.social and @benmorehouse.bsky.social labs that were so instrumental to this project! A special thank you to our collaborators L. Aravind and Max Burroughs for their incredible bioinformatic analyses!