Journal of Infection and Public Health
A novel
By Hanyu Fu, Zihe Zhou, Zhiheng Tang and 7 others.
Abstract
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.