Bacteriophage antidefense genes that neutralize TIR and STING immune responses

Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as a central immune defense strategy to prevent the spread of bacteriophage viruses and other invasive genetic elements across a population. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to rapidly mobilize cognate death effectors. Here, we identify a large family of bacteriophage nucleotidyltransferases (NTases) which synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit NAD-depleting TIR effectors activated through a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of host TIR-STING suicide signaling. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphatase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a role for the alarmone-activated host toxin MazF as a key executioner of TIR-driven abortive infection. Phage NTases and counter-defenses like Atd1 preserve host viability to ensure virus propagation, and may be exploited as tools to modulate TIR and STING immune responses. Overall design: The pooled phage ORF library was transformed into BL21 (DE3) Rosetta 2 competent cells (MilliporeSigma) together with pET30a encoding Flag-TIR-STING or empty vector (EV) control. 100 ml of overnight cultures were diluted 1:20 and grown in M9 media supplemented with 0.25% CAA and 1% glycerol, and induced at A600=0.3-0.4 through the addition of 0.5mM IPTG. Samples were taken immediately prior to induction and at 18 h post-induction. Reads were obtained from DNA isolated from cultures containing EV or TIR-STING, at either time 0 or after 18 hr induction.