Dams, D.; Pas, C.; Latka, A.; Drulis-Kawa, Z.; Fieseler, L.; Briers, Y.

Phage tail-like bacteriocins, or tailocins, provide a competitive advantage to producer cells by killing closely related bacteria. Morphologically similar to headless phages, their narrow target specificity is determined by receptor-binding proteins (RBPs). While RBP engineering has been used to alter the host range of a selected R2 tailocin from Pseudomonas aeruginosa, the process is labor-intensive, limiting broader application. We introduce a VersaTile-driven R2 tailocin engineering platform to scale up RBP grafting. This platform achieved three key milestones: (1) engineering R2 tailocins specific to Escherichia coli serogroups O26, O103, O104, O111, O145, O146 and O157; (2) reprogramming R2 tailocins to target for the first time capsule and a new species, specifically the capsular serotype K1 of E. coli and K11 and K63 of Klebsiella pneumoniae; (3) creating the first bivalent tailocin with a branched RBP and cross-species activity, effective against both E. coli K1 and K. pneumoniae K11. Over 90% of engineered tailocins were effective, with clear pathways for further optimization identified.