A VersaTile Approach to Reprogram the Specificity of the R2-Type Tailocin Towards Different Serotypes of and

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 target range of a selected R2 tailocin from , the process is labor-intensive, limiting broader application. We introduce a VersaTile-driven R2 tailocin engineering and screening platform to scale up RBP grafting. This platform achieved three key milestones: (I) engineering R2 tailocins specific to serogroups O26, O103, O104, O111, O145, O146, and O157; (II) reprogramming R2 tailocins to target, for the first time, the capsule and a new species, specifically the capsular serotype K1 of and K11 and K63 of ; (III) creating the first bivalent tailocin with a branched RBP and cross-species activity, effective against both K1 and K11. Over 90% of engineered tailocins were effective, with clear pathways for further optimization identified. This work lays the groundwork for a scalable platform for the development of engineered tailocins, marking an important step towards making R2 tailocins a practical therapeutic tool for targeted bacterial infections.