Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis

The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic “phagebodies.” We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications. [Display omitted] •Vastly diverse phagebody libraries containing 107 different members were created•Structure-informed engineering of viral tail fibers generated host-range alterations•Bacterial resistance to phagebodies was not observed across long timescales•Select phagebodies limit bacterial growth in a mouse wound infection model Bacteriophage libraries containing millions of variants of phage tail fiber motifs on a common structural scaffold give rise to infectious phages with expanded or altered host ranges, which may be useful for phage therapy efforts.