Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.

The evolutionary arms race between bacteria and bacteriophages drives rapid evolution of bacterial defense mechanisms with scattered distribution across genomes. We hypothesized that this variability in bacterial defense systems leads to equally variable counter-defense repertoires in phage genomes. Examining the variable regions in Pseudomonas model phages of the Pbunavirus genus revealed five anti-defense genes, including one inhibiting Druantia type III named DadIII-1, another targeting Thoeris type III named TadIII-1, one inhibiting Zorya type I named ZadI-1, and two related broad defense inhibitors named Bdi1 and Bdi2 targeting four defenses. A typical Pbunavirus encodes up to five known anti-defense genes, some inhibiting four unrelated defense systems with distinct nucleic-acid-targeting mechanisms. Structural homologs of broad-acting Bdi1 and Bdi2 are encoded across diverse phage taxa infecting multiple bacterial hosts. These findings show that phages face a variety of bacterial defenses, driving them to evolve both specific and general strategies to overcome these barriers.