Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence

cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence 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 signalling systems (CBASS) have been discovered(8-11). These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% contain Cap2 and Cap3, which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively(8,12). Although these proteins are required to prevent infection of some bacteriophages(8), the mechanism by which the enzymatic activities exert an 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 increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.

Automated summary

cGAS is a conserved enzyme that plays a crucial role in immune defense. It is activated by DNA to produce cGAMP, which leads to the expression of antimicrobial genes. In bacteria, a similar enzyme system called CBASS has been discovered, where a protein called Cap2 forms a thioester bond with cGAS and promotes its conjugation to target proteins, increasing cGAMP production.