Mechanisms of cyclic-oligonucleotide-based anti-phage signalling systems in prokaryotic immunity

Cyclic oligonucleotide based anti-phage signalling systems (CBASS) are a diverse form of prokaryotic immunity, characterised by a core nucleotide cyclase and effector pair that, upon infection, produce secondary messengers and activate cell disrupting phenotypes, preventing phage replication. CBASS cyclases share ancestry with the eukaryotic innate immune pathway, cGAS-STING, and produce a diverse array of cyclic nucleotide molecules. This has both rapidly expanded the known arsenal of prokaryotic defence signals and identified a mechanism of immunity conserved across all domains of life. Whilst the CBASS cyclase/effector pair alone offers immunity against select phage, diverse ancillary protein architectures are observed within CBASS to facilitate broader phage protection. Type II CBASS is characterised by the presence of ancillary proteins Cap2 and Cap3, which share homology with eukaryotic ubiquitin conjugation and deconjugation machinery, respectively. Type II ancillary genes have been demonstrated as essential for broad CBASS protection in vivo and were predicted to regulate the cyclase during infection, potentially through ubiquitin-like conjugation and post-translational modification. However, their critical roles within CBASS activation and regulation remained elusive. During this project focussed on the type II CBASS system from Bacillus cereus, we determined that the monomeric wild-type cyclase adopts a low-activity conformation, subject to significant inhibition at cellularly relevant nucleotide concentrations. Following identification of the cyclase as the Cap2 conjugation substrate, we identified several major conjugation targets, including Phage shock protein A (PspA) and other cyclase monomers during different stages of infection. Prior to infection, the cyclase is spatially organised through conjugation to PspA, whilst cyclase homo-oligomerisation likely functions to structurally rearrange the cyclase active site, forming active oligomeric species. Additionally, we have elucidated that Cap2 conjugation is directly reversed by Cap3, facilitating re- conjugation and enhancing Cap2 conjugation target specificity through rapid non- target conjugate cleavage.