Characterization of a K62-specific phage depolymerase and its protective effect in a mouse model of Klebsiella pneumoniae infection.

Abstract

Multidrug-resistantKlebsiella pneumoniae has emerged as a formidable healthcare challenge, driving the urgent need for alternative therapeutic strategies such as phage therapy. In this study, we isolated and comprehensively characterized a novel phage, designated vB_Kp_IME328 (IME328), which exhibits highly specific bactericidal activity against K62-type K. pneumoniae. Morphological analyses revealed that IME328 forms plaques surrounded by distinct translucent halos and possesses a long tail, clustering it within the Webervirus genus. Through whole-genome sequencing and bioinformatic analysis, we identified ORF3 as the gene encoding a putative tail-fiber depolymerase. This gene was successfully cloned and expressed, yielding the recombinant protein Dp3. Functional assays confirmed that Dp3 possesses robust depolymerizing and antibacterial activities. In vitro, Dp3 acted synergistically with serum to effectively eradicate the target bacteria. Furthermore, live/dead cell staining and cellular assays demonstrated that Dp3 significantly enhances the phagocytic and intracellular bactericidal capacities of macrophages. In vivo evaluations using a lethal murine bacteremia model (inoculated with 6 × 10⁷ CFU) showed that Dp3 administration conferred 100% protection and survival, whereas all untreated infected mice succumbed to the infection. Together, our findings highlight the targeted anti-virulence properties of the depolymerase Dp3. By enhancing host-mediated immune clearance, Dp3 offers a promising therapeutic strategy against antibiotic-resistant K62-type K. pneumoniae infections.