From structure to function: binding determinants of a miniaturised phage depolymerase and their immunological relevance in Klebsiella pneumoniae capsule degradation.

The global rise of multidrug-resistant bacteria is a major health threat, with Klebsiella pneumoniae identified by the WHO as a critical priority pathogen. Phage-derived depolymerases have emerged as promising countermeasures because they can degrade the capsular polysaccharide (CPS) that shields bacteria from the immune system, thereby increasing their susceptibility to antibiotics and host defences. However, the limited understanding of how they recognise specific CPS structures remains a major obstacle to developing effective depolymerase-based therapeutics. In this study, we provide a comprehensive NMR and mutational analysis to characterise the mechanism of action of a miniaturised depolymerase, here mKP34gp57, targeting the clinically relevant K63 CPS. We show by NMR that mKP34gp57 hydrolyses CPS with high efficiency through an endoglycosidase-retaining mechanism. During substrate recognition, our data demonstrate that the enzyme interacts predominantly with the galactose and fucose moieties, which serve as the critical recognition features and thus the principal determinants of CPS specificity. Computational studies provide structural clues for the roles of the catalytic residues E266/E300 and D151. Finally, we prove that the hexasaccharide produced upon CPS hydrolysis stimulates dendritic cell maturation and T-helper-driven lymphocyte proliferation. Identifying the binding determinants that govern CPS recognition by mKP34gp57, and using this information to generate immunogenic fragments, deepens our understanding of how minidepolymerases can be rationally engineered to achieve tailored serotype specificity, and improved therapeutic and diagnostic potential.