Native pathogen-phage networks reveal multilayered defense and gut-active anti-phage immunity.

Bacteriophages drive pathogen evolution, yet the mechanisms governing phage susceptibility in native bacterial backgrounds, and their consequences within mammalian hosts, remain poorly understood. Here, we mapped >1,000 interactions between 20 diverse Salmonella isolates and 52 wild phages from global disease reservoirs, integrating genome-scale fitness profiling with comparative genomics. Receptor identity and surface phase variation, including Hin-mediated flagellar switching, explained ∼67% of phage susceptibility patterns, identifying cell-surface architecture as the major determinant of phage host range in Salmonella. Among resistance phenotypes not explained by surface features, we discovered anti-phage-packaging agent (AppA), a prophage-encoded defense factor that abrogates phage replication within its native host. AppA inhibits phage DNA packaging through functional mimicry of a terminase assembly interface, revealing a previously unrecognized mechanism of phage defense. AppA is expressed under conditions encountered during mammalian infection and suppresses phage expansion in the murine gut, demonstrating that prophage-encoded single-gene defenses can shape disease outcomes in vivo.