Thank you Jens!

And you started it all!

Thank you Sadie!

Huge thanks to co-first author Azita Leavitt - as always, it was so fun and fruitful to work together! Special thanks also to @bhurieva.bsky.social, Alla H. Falkovich, @nbechon.bsky.social, @francoisrousset.bsky.social, @ostermanilya.bsky.social, and @soreklab.bsky.social !

Our work expands the landscape of TIR immune signaling in microbes and reveals unexpected connections with plant and animal immunity. Many Thoeris effectors and signals remain uncharacterized, and uncovering them could once again reshape our understanding of immunity across life🛡️🦠🌱🧍‍♀️

We found that Thoeris immunity is far more common than previously thought. Thoeris systems appear in ~8% of microbial genomes, about twice what was previously estimated! We also found that ~20% of microbes encode at least one signaling system: Thoeris, CBASS, or Pycsar.

Type VII Thoeris uses a SLATT-domain effector (ThsH), likely forming membrane-breaching oligomers upon cADPR binding, while type VIII encodes a CMP-hydrolase effector (ThsI) predicted to disrupt nucleotide pools to abort phage infection.

Two Thoeris types (types VII and VIII) use canonical cADPR as their immune signal, a molecule well known in human immunity but not previously seen in bacteria. These cADPR-based systems are widespread, revealing a surprising evolutionary link to human immune signaling.

Adding 2′cADPR to cells expressing type VI systems triggered effector-mediated toxicity, and phages engineered to express 2′cADPR-sequestering sponge proteins overcame defense, confirming the signal’s identity!

Most new Thoeris systems did not produce any known Thoeris signals. LC-MS revealed that TIR proteins from type VI Thoeris synthesize 2′cADPR upon phage infection, the same signal broadly used by plant immune TIRs, to activate their cognate effector ThsG.

Structural modeling shows that ThsF forms a membrane-spanning homo-heptamer with positively charged pockets that likely bind 3′cADPR, leading to membrane disruption.

Type V Thoeris detects phage infection and produces the immune signal 3′cADPR (the same signal used by type I). This molecule activates the DUF2270 effector ThsF, whose toxicity is triggered specifically by 3′cADPR and not by related cADPR isomers.

To identify the immune signals of the systems, we expressed each TIR alone, infected with phage, and collected lysates to capture the TIR-derived molecules. We then characterized these signals using LC-MS and in vitro assays that test activation of signal-specific effectors.

We experimentally tested and validated Thoeris operons, expanding Thoeris immunity from four to eleven types (I–XI)!

We found Thoeris effectors ranging from DNases and RNases to phospholipases, ion channels, transmembrane proteins, and many DUFs! Several of these also appear as cell-killing effectors in other defense systems, strongly supporting their roles as new Thoeris effectors.

By analyzing ~600 million microbial proteins from genomes and metagenomes, we mapped the full landscape of TIR-based immune signaling in bacteria and archaea. We identified >10,000 candidate Thoeris systems linked to proteins not previously recognized as Thoeris effectors.

Four Thoeris types were known, with three characterized, each relying on a different TIR-derived signal and effector protein to block phage infection.

TIR domains are ancient immune components found across the tree of life. In bacteria, they form the core of Thoeris, an antiviral system in which a TIR protein senses phage attack, produces an ADPR-derived immune signal, and activates effector proteins that block infection.