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-{beta}-lactamase (MBL)-fold hydrolase, /{beta}-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 /{beta}-hydrolase are zymogens activated by cleavage at two distinct sites, and upon proteolysis, MBL becomes a Zn2+-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.