The virulent phage vB_KpnP_JYSS5 and its depolymerase Dep11 targeting K57 Klebsiella pneumoniae: characterization and therapeutic potential against biofilm and systemic infection

Abstract

Klebsiella pneumoniae frequently causes hospital-acquired and community-acquired infections, posing a serious threat to human health. Lytic phages can rapidly propagate within host bacterial cells and release progeny phages, ultimately inducing the death of the host bacteria. K. pneumoniae depolymerases are enzymes capable of specifically degrading bacterial surface polysaccharides, thereby disrupting bacterial biofilms, which offers a new approach to the prevention and control of K. pneumoniae . Morphology of phage vB_KpnP_JYSS5 was observed through transmission electron microscopy. The optimal multiplicity of infection, adsorption curve, one-step growth curve, and temperature and pH stability were determined using the double-layer agar plate method. Whole-genome sequencing was performed on the DNBSEQ-T7 sequencing platform. The putative depolymerase gene (ORF11) from the phage genome was subjected to prokaryotic expression. The anti-biofilm activity of the phage and the depolymerase was assessed using plate counting, scanning electron microscopy, and confocal laser scanning microscopy. The effects of depolymerase treatment on macrophage phagocytic function were evaluated using plate counting, ELISA, and flow cytometry. A mouse infection model was established by intraperitoneal injection of K. pneumoniae to assess the efficacy of the depolymerase. Phage vB_KpnP_JYSS5 specifically lyses the K57-type K. pneumoniae strain 21AA3004. It is a lytic phage with a DNA genome of 40,795 bp. The optimum MOI is 0.001. It has an average burst size of approximately 114 PFU/cell. The phage retained its infectivity between 4 °C and 60 °C and at a pH range of 5 to 11. The phage effectively inhibited biofilm formation and degraded pre-existing biofilms of host strain. Encoded by ORF11, Dep11 effectively degraded the capsule, suppressed biofilm formation, and eliminated mature biofilms but also potentiated polymyxin B and gentamicin against bacteria. Dep11 enhanced macrophage phagocytosis. In animal experiments, Dep11 (50 µg) raised the survival rate of infected mice to 70% and lowered four pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ) in their lung tissue. Phage vB_KpnP_JYSS5 is a novel double-stranded DNA phage classified within the order Autographivirales . Depolymerase Dep11 specifically targets the capsular polysaccharide of K57 K. pneumoniae , making it a promising therapeutic agent for the treatment of K57 K. pneumoniae infections.