A VersaTile approach to reprogram the specificity of the R2-type tailocin towards different serotypes of Escherichia coli and Klebsiella pneumoniae

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. ImportanceWhile tailocin engineering is a proven and promising concept, the current engineering approach lacks scalability, limiting a vast exploration. This study advances tailocin engineering by increasing its throughput. Implementing a scaled up approach, we have shown the flexibility of the R2 tailocin scaffold to accommodate diverse receptor-binding domains, expanding its functionality to target a new type of receptor (capsule) and a previously untargeted species. In addition, functional tailocins with branched receptor-binding proteins portraying dual, cross-genus activity were produced. 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.