Ubiquitin-like Conjugation by Bacterial cGAS Enhances Anti-phage Defence.

cGAS is an evolutionarily conserved enzyme that plays a pivotal role in immune defense against infection 1-3 . In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP-AMP (cGAMP) 4,5 , which leads to the expression of antimicrobial genes 6,7 . In bacteria, cyclic dinucleotide (CDN)-based anti-phage signaling systems (CBASS) have been discovered 8-11 . These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria 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, respectively 8,12 . Although these proteins are required to prevent infection of some bacteriophages 8 , 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 cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increased the production of cGAMP. Through a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signaling by binding tightly to cGAMP (Kd ~30 nM) and sequestering 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 CDN levels.