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. 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.