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.

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)!

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.

The phage-derived CBASS inhibitor can also bind the human cGAS immune enzyme and block its signaling activity, demonstrating once again that phage genomes are a rich reservoir of immune modulators capable of antagonizing bacterial, plant, and animal immunity 🦠🌿🧍‍♂️

Strikingly, one of the Thoeris inhibitors also binds and inhibits distantly related TIR domain proteins in humans and plants, suggesting that conservation between prokaryotic and eukaryotic immune proteins renders the eukaryotic homologs susceptible to phage-encoded inhibitors!

Acb3 neutralizes CD-NTase enzymes by wrapping around the immune protein, 'hugging' it tightly, and inserting a loop into its active site - blocking access to active sites that produce or recognize immune signals emerges as a common viral strategy for inhibiting signaling systems

The sixth family of anti-Thoeris proteins inserts decoy loops into the signal recognition site of the Thoeris effector and interacts with key residues that would otherwise bind the Thoeris immune signal

In the bacterial defense system Thoeris, infection stimulates TIR domains to produce an immune signaling molecule. We identified five families of anti-Thoeris proteins that bind Thoeris TIR proteins and insert flexible loops into their active sites to block signaling activity 🚫

We developed a computational and experimental pipeline that utilizes iterative structure predictions to analyze over 30M phage-encoded proteins for inhibitors that physically bind and antagonize host immune proteins ⚔️🛡️