Balancing of immune activation and suppression during phage infection

Abstract Signaling-based anti-bacteriophage systems such as CBASS and Thoeris synthesize infection-triggered nucleotide signals that activate anti-phage effectors 1,2 . However, the phage features sensed by these systems and the mechanisms phages use to evade signaling immunity remain poorly understood. Here, studying clinically relevant Pseudomonas aeruginosa phages from the Migulavirinae family 3 , we show that closely related phages encode subtle allelic variation in side tail fiber proteins that determine sensitivity to type II Thoeris. In parallel, these same phages encode an “anti-defense hotspot” that contains three adjacent genes that are each sufficient to facilitate phage evasion of both CBASS and Thoeris defenses, counter-balancing the activating proteins. Comparative analysis of this anti-signaling hotspot across the broader family of related N4-like phages uncovered a new Thoeris anti-defense (Tad) protein that sponges NAD-derived molecules (e.g. gcADPR) and exhibits sequence and structural similarity to a poorly characterized nucleotide-binding region of the human ryanodine receptor. Together, these findings reveal how the balance between immune activation and antagonism shifts phage outcomes and reveals a surprising similarity between a phage molecular sponge and an important human protein.