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.

Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes.

Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains.