UCSF

A fundamental strategy of eukaryotic anti-viral innate immunity involves the cGAS enzyme, which synthesizes 2’,3’-cGAMP and activates a STING effector to limit viral replication. Bacteria contain cGAS-like enzymes producing a diversity of cyclic oligonucleotide signals that activate an effector protein to induce cell death upon bacteriophage (phage) infection, known as CBASS. It remains unknown whether bacteria endogenously expressing CBASS exerts anti-phage activity and how these bacteria and phage co-evolve together. Here, we identified endogenously active Type II-A CBASS in Pseudomonas aeruginosa that contains a cGAS-like enzyme (CdnA) producing 3’,3’-cGAMP, which signals to a phospholipase (CapV) effector and limits dsDNA lytic and temperate phage replication >10,000-fold. In response, phages evolved an anti-CBASS protein (Acb2) that forms a hexamer with three cGAMP molecules to ‘sponge’ up signals and effectively inhibit both bacterial CapV and eukaryotic STING effector activity. Excitingly, Acb2 binds to an even broader spectrum of CBASS cyclic dinucleotides (CDNs), like 3’,3’-cUU/UA/UG, and cyclic trinucleotides (CTNs), like 3’,3’,3’-cAAA/cAAG. One Acb2 hexamer can independently and simultaneously bind three CDNs and two CTNs, enabling phages to inhibit bacteria with Type II-A (3’,3’c-cGAMP-producing) and Type III-C (cA3-producing) CBASS. These collective findings establish a new paradigm of viral proteins that sponge a remarkable breath of cyclic oligonucleotide molecules and inhibit signaling-based immunity across all domains of life. Upon deletion of Acb2, phage mutants selected under CBASS immune pressure were forced to evolve mutations in their major capsid protein to escape. While there is growing evidence of phages mutating essential structural proteins to escape CBASS, the reason for evasion remains unclear. Despite this, to counteract CBASS resistant phages, we observed that increasing CdnA enzymatic activity and 3’,3’-cGAMP production vastly increases CBASS anti-phage activity and overcomes phages expressing Acb2 or mutant capsids. Surprisingly, counter to the established dogma that CBASS induces cell death, the hyperactive and endogenously active Type II-A CBASS in P. aeruginosa induces cell growth and protects bacteria from phage-induced lysis. Altogether, these findings demonstrate that native bacterial hosts exert CBASS immunity and that phages can evolve and counteract this immune response through potent inhibitors that ‘sponge’ CBASS signaling molecules or acquisition of mutations in structural proteins.