Proteolytic activation of diverse antiviral defense modules in prokaryotes

Linked protease–effector modules are widespread in prokaryotic antiviral defense, yet the mechanisms of most remain poorly understood. Here we show that four of the most prevalent modules—metallo-β-lactamase (MBL)-fold hydrolase, α/β-hydrolase, Pepco, and EACC1—form latent death effectors that are unleashed by site-specific proteolysis. Genetic, biochemical, and structural analyses reveal novel modes of effector licensing. MBL and α/β-hydrolase are zymogens activated by cleavage at two distinct sites, and upon proteolysis, MBL becomes a Zn 2+ -dependent double-stranded DNA nuclease. In contrast, Pepco and EACC1 act as pore-forming toxins via distinct mechanisms: Pepco constitutively oligomerizes into a denaturation-resistant beta barrel that is activated by cleavage after a specific isoleucine in its C-terminal tail, whereas EACC1 monomers assemble into large membrane pores following removal of an autoinhibitory domain. All four modules are fused to diverse sensors to detect a wide range of phage signals, and EACC1–DnaK chaperone fusions suggest a convergence between defense and general stress responses. These findings establish proteolysisgated activation as a dominant, modular logic for anti-phage defense and reveal parallels with eukaryotic innate immunity.