Mapping Structural Constraints and Adaptive Potential in a Capsule-Degrading Phage Tailspike Protein.

Bacteriophage tailspike proteins (TSPs) degrade bacterial capsules to enable infection, yet the molecular determinants of their function and host range remain unclear. We applied deep mutational scanning (DMS) to the endosialidase TSP of Escherichia coli K1 phage K1F, generating 22,365 single-amino-acid variants using an enhanced ORACLE phage engineering platform. Functional scores revealed the TSP is structurally fragile yet harbors pockets of adaptive flexibility. Mutations within the β-propeller active site uncovered residues accommodating longer sialic acid chains than captured by structural studies, while the β-helix stalk emerged as an adaptive "tuning knob" modulating processivity and specificity. Comparative selections across K1 strains identified discrimination hotspots in β-barrel loops and distal residues outside canonical binding sites, implicating capsule modifications and O-antigen presence as key modulators of host range. By resolving how specific mutations modulate function and host range, this study offers a roadmap for designing phages that overcome capsule-based defenses in pathogenic bacteria.