A novel Przondovirus bacteriophage combats Klebsiella pneumoniae through evolutionary trade-off and metabolic reprogramming

BACKGROUND: Bacteriophages represent a promising alternative for combating carbapenem-resistant hypervirulent Klebsiella pneumoniae (Kp). However, the molecular mechanisms underlying phage-host co-evolution and the action of phage-derived anti-virulence effectors remain insufficiently explored. We hypothesized that a novel Przondovirus phage could suppress Kp virulence through receptor-mediated evolutionary trade-offs and disruption of metabolic pathways. METHODS: A novel phage, P1524, was isolated from hospital sewage and characterized using transmission electron microscopy, one-step growth curve analysis, and stability testing. Its host range was evaluated against 86 clinical Kp isolates. Phage-resistant mutants were generated and analyzed through whole-genome sequencing. Three phage-encoded effectors (Orf4, Orf15, Orf23) were cloned and expressed in Kp, followed by assessments of bacterial growth, biofilm formation, serum resistance, and in vivo survival in a Galleria mellonella model. Quantitative proteomics and quantitative real-time polymerase chain reaction (RT-qPCR) were conducted to identify host metabolic pathways targeted by these effectors. RESULTS: P1524 exhibited broad lytic activity against high-risk Kp clones (ST11, ST14, ST65). Resistance to P1524 was attributed to a wcaJ missense mutation (L365I/P366R), which induced an evolutionary trade-off characterized by capsule loss, impaired biofilm formation, and reduced in vivo virulence. Three novel phage effectors-Orf4, Orf15, and Orf23-significantly enhanced survival in Galleria mellonella models. Mechanistically, Orf4 and Orf23 downregulated key enzymes of the 2,3-butanediol fermentation pathway (AldB_1, AlsS, and BudC) at both protein and transcriptional levels, effectively blocking this metabolic route. CONCLUSIONS: The Przondovirus phage P1524 combats Kp through a wcaJ mutation-mediated evolutionary trade-off and metabolic reprogramming via the 2,3-butanediol fermentation pathway. These findings underscore the potential of phage-derived metabolic inhibitors as innovative antimicrobial agents.