Ubiquitin-Like Conjugation by Bacterial cGAS Enhances Anti-Phage Defense

Abstract cGAS is an evolutionarily conserved enzyme that plays a pivotal role in immune defense against microbial infection1-3. In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP-AMP (cGAMP)4,5 , which initiates a signaling cascade leading to the expression of a battery of immune defense genes6,7. In bacteria, cyclic dinucleotide (CDN)-based anti-phage signaling systems (CBASS) have been discovered8-11. These systems are composed of cGAS-like enzymes and various effector proteins that either kill bacteria or inhibit bacterial growth upon phage infection, thereby stopping phage spread. Of the CBASS systems reported, ~39% contain Cap2 and Cap3, which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating (DUB) enzymes, respectively8,12. Although these proteins have been shown to be required to prevent infection of some bacteriophages8, the mechanism by which the enzymatic activities exert anti-phage effect is unknown. Here, we show that Cap2 forms a thioester bond with the C-terminal glycine of bacterial cGAS and promote conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. Mutations of the C-terminal glycine of cGAS abrogated its anti-phage activity in several CBASS systems, recapitulating the effect of mutating the active site cysteines of E1- and E2- like domains in Cap2. The covalent conjugation of cGAS increased the production of cGAMP. Through a genetic screen with mutagenesis of T4 phage, we identified the phage protein Vs.4. that antagonizes the effect of cGAS conjugation catalyzed by Cap2. Vs.4 bound to cGAMP with a Kd of ~30 nM and sequestered it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrate an arms race between bacteria and viruses through controlling the levels of CDNs.