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.
26The rapid emergence of antibiotic-resistant infections is prompting increased interest in 27 phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the 28 successful development of phage therapies. Through natural evolution and structural modeling, 29 we identified host-range determining regions (HRDR) in the T3 phage tail fiber protein and 30 developed a high-throughput strategy to genetically engineer these regions through site-directed 31 mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional 32 diversity (>10 7 different members), while minimizing disruptions to the overall protein structure, 33 resulting in synthetic "phagebodies". We showed that mutating HRDRs yields phagebodies with 34 altered host-ranges. Select phagebodies enable long-term suppression of bacterial growth by 35 preventing the appearance of resistance in vitro and are functional in vivo using a mouse skin 36 infection model. We anticipate this approach may facilitate the creation of next-generation 37 antimicrobials that slow resistance development and could be extended to other viral scaffolds for 38 a broad range of applications. 39 evolution, tail fiber 41 3 Highlights: 42 Vastly diverse phagebody libraries containing 10 7 different members were created. 43 Structure-informed engineering of viral tail fibers efficiently generated host-range 44 alterations. 45 Phagebodies prevented the development of bacterial resistance across long timescales in 46 vitro and are functional in vivo. 47 48 49 59 Schooley et al., 2017). In addition, phages are selective for particular bacterial strains, as opposed 60 conventional chemical antibiotics that exhibit broad-spectrum activity, which contributes to 61 antibiotic-resistance development. Phage selectivity is dependent on binding to cell surface 62 receptors in order to recognize their host and initiate infection (Silva et al., 2016). However, 63 4reliance on receptor recognition for infectivity implies that resistance against a bacteriophage can 64 occur through receptor mutations. As a result, phage-based products are usually composed of 65 multiple unrelated phages that collectively target a range of receptors, thus distributing the 66 selective pressure away from any individual phage receptor. However, these cocktails are often 67 composed of uncharacterized phages whose biology are poorly defined. Furthermore, 68 manufacturing cocktails composed of diverse phages, as well as tracking their pharmacodynamics 69 and immunogenic properties, is complex, which can limit their development as antimicrobial drugs 70 (Cooper et al., 2016). 71 Various approaches have been undertaken to rationally expand the host-range of phages to 72 combat resistance (74 2005; Yosef et al., 2017). However, these approaches depend on hybridization between already 75 characterized bacteriophages with known and desired host-ranges. Natural evolution of phages has 76 also been harnessed to alter phage host-range, but...
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