Two tail protein-derived capsule depolymerases from phage vB_KpnS_GH-K1 are effective against pneumonia caused by K2 serotype Klebsiella pneumoniae.

Abstract

OBJECTIVES: Hypervirulent Klebsiella pneumoniae (hvKP) poses an escalating threat to public health and animal husbandry, underscoring the urgent need for novel antimicrobial alternatives. This study aimed to isolate and characterize depolymerases from a Klebsiella phage and evaluate their therapeutic potential against acute pneumonia caused by the K2 serotype hvKP METHODS: We isolated and characterized the bacteriophage vB_KpnS_GH-K1 (GH-K1) of the "KP36likevirus" genus. Genomic analysis led to the identification of two putative phage-derived depolymerases: tail fiber protein (TFP41) and tail spike protein (TSP42). The enzymatic activity of the expressed and purified proteins was assessed through spot assays and mucoviscosity measurements. Their ability to enhance host immune clearance was evaluated via macrophage phagocytosis assays, analysis of mitogen-activated protein kinase (MAPK) signaling pathway activation, and cytokine profiling. The therapeutic efficacy was ultimately determined in a murine model of acute pneumonia, where survival rates, lung bacterial loads, and pathological changes were monitored following intranasal administration of the depolymerases. RESULTS: Both depolymerases exhibited potent activity in degrading the capsular polysaccharides (CPS) of K2 serotype K. pneumoniae. Capsule degradation by TFP41 and TSP42 enhances macrophage-mediated phagocytosis of K2 serotype strains, leading to potentiated activation of the MAPK signaling pathway. In vivo experiments demonstrated the exceptional therapeutic efficacy of TSP42, where a single 20 µg dose ensured over 80 % survival in mice within 12 h of intranasal infection, achieving complete survival when administered within 2 h. Importantly, TSP42 demonstrates superior therapeutic efficacy against acute pneumonia caused by K2 strains compared to other depolymerases in "KP36likevirus". Although TFP41 exhibited slightly weaker efficacy, daily administration for three consecutive days also resulted in 100 % survival, and notably represents the first tail fiber depolymerase with demonstrated therapeutic efficacy in the "KP36likevirus" genus, expanding the functional diversity of phage-derived depolymerases beyond well-characterized tail spike proteins. CONCLUSION: These findings underscore the potential of phage-derived depolymerases as innovative therapeutic agents against hvKP infections and position TSP42 as an exceptionally promising antibiotic alternative.