Phage-derived depolymerase targeting K27 capsule impairs <i>Klebsiella pneumoniae</i> virulence, biofilm formation, and promotes immune clearance

The global rise of multidrug-resistant <i>Klebsiella pneumoniae</i> underscores the urgent need for alternative therapeutic strategies. Bacteriophage-derived depolymerases have emerged as promising antimicrobial factors, selectively degrading bacterial capsules and impairing key pathogenic traits. We characterize a novel depolymerase, PRA33gp45, associated with the structural protein of bacteriophage vB_KpnP_PRA33. Bioinformatic structural analyses predicted endo-N-acetyl neuraminidase-like activity and canonical depolymerase domain architecture. The recombinant PRA33gp45 specifically hydrolysed capsular polysaccharides (CPS) of K27-serotype <i>K. pneumoniae</i> and produced characteristic halo zones on bacterial lawns, confirming its enzymatic activity. Capsule staining demonstrated rapid and progressive capsule degradation within 120 minutes of treatment. PRA33gp45 significantly inhibited biofilm formation, disrupted mature biofilms, and altered biofilm architecture as visualized by confocal microscopy. Depolymerase pre-treatment markedly reduced <i>K. pneumoniae</i> survival within A549 human lung epithelial cells, without exhibiting any cytotoxic effect and sensitized bacteria to complement-mediated killing in human serum. Finally, PRA33gp45 treatment of <i>K. pneumoniae</i> lowers morbidity and mortality in the <i>Galleria mellonella</i> larvae model. Collectively, these findings identify PRA33gp45 as a novel and highly specific depolymerase that diminishes <i>K. pneumoniae</i> virulence by targeting its protective capsule, impairing persistence as biofilm, and enhancing innate immune clearance. Its safety and efficacy suggest potential as an antimicrobial or adjuvant therapeutic agent against K27-type <i>K. pneumoniae</i> infections, particularly in the context of multidrug resistance and emerging pathogens.