Project Summary Bacteriophages (phages) are viruses that infect and kill bacteria. These viral predators have provided the selective pressure to drive the evolution of numerous anti-phage immune pathways such as restriction- modification and CRISPR-Cas systems. A recent surge in the discovery of new anti-phage immune systems has uncovered an incredible diversity of defensive mechanisms across prokaryotes. Strikingly, many of these systems appear to be homologous to mammalian anti-viral immune pathways, suggesting that some human innate immune responses may have originated as anti-phage systems. The immune system of interest in this proposal first arose in bacteria and is now widely studied in mammals as cGAS-STING. In mammals, the cGAS enzyme directly binds to cytoplasmic double-stranded DNA (dsDNA) and triggers the production of cyclic GMP- AMP (cGAMP) molecules that bind to the protein STING to ultimately stimulate interferon genes. cGAS is normally held in the off state through a wide array of inhibitory post-translational modifications, direct protein interactions, nucleosome tethering, and phase separation. However, the regulatory mechanisms that inhibit or activate the homologous system in bacteria, called CBASS, remain unknown. CBASS (cyclic oligonucleotide-based anti-phage signaling systems) systems are currently thought to drive an abortive infection outcome, in which the production of one of many potential cyclic oligonucleotides (c-oligos) activates a co- encoded toxic effector protein and induces cell death. Given this cell death outcome of activated CBASS, we hypothesize that tight regulatory mechanisms must keep it off, and these mechanisms must be rapidly reversed during phage infection to turn CBASS on. Biochemical assays have shown that the cGAS-like enzymes in bacteria, called CD-NTases, constitutively produce c-oligos in vitro and structural work shows that CD-NTases are in an activated state with the catalytic site permanently competent for substrate nucleotide binding. Paradoxically, the overexpression of the CD-NTase and effector in bacteria is not toxic in the absence of phage, confirming that the cell has at least one mechanism to repress or inhibit function. For our studies, we will use the first described native model system for CBASS anti-phage function, established in Pseudomonas aeruginosa, in our group. We will first conduct unbiased and targeted genetic screens to identify endogenous CBASS inhibitors or repressors that allow maintenance and prevent self-toxicity by CBASS. In conjunction, we will identify the phage component(s) that triggers CBASS by isolating phages that acquire mutations that enable CBASS escape. Genetics and biochemical experiments will be used to validate the trigger. Surprisingly, preliminary experiments with this approach revealed the first phage encoded anti-CBASS protein, which will be mechanistically characterized during this study. Together, these experiments will provide a mechanistic unde...