Enhancing the Antibacterial Activity of Endolysin against Klebsiella pneumoniae through Fusion Engineering Using Antimicrobial Peptide Sub5.

The majority of clinically isolated Klebsiella pneumoniae (K. pneumoniae) strains are opportunistic pathogens exhibiting multidrug resistance (MDR), which has driven the exploration of alternative therapeutic approaches. Endolysins, as phage-derived lytic enzymes, represent highly promising novel antimicrobial agents. However, their application against Gram-negative bacteria is constrained by the physical barrier of the outer membrane (OM). Therefore, conferring the ability for endolysins to penetrate or disrupt the OM, thereby reaching the target peptidoglycan, is crucial. In this study, we fused the antimicrobial peptide (AMP) Sub5 with LysK1, a phage-derived endolysin targeting K. pneumoniae. Leveraging Sub5's membrane-targeting mechanism, we enabled LysK1 to traverse the OM barrier of K. pneumoniae, targeting the peptidoglycan layer to exert synergistic antimicrobial effects. The results showed that, compared with LysK1, LysK1-Sub5 reduced the minimum inhibitory concentration (MIC) against K. pneumoniae by 2.7 to 6.1-fold and was able to inhibit the growth of K. pneumoniae within 16 h without the need for an OM permeabilizer. In addition, it exhibited stable environmental tolerance and broad-spectrum lytic activity. In vivo experiments using a mouse skin wound infection model demonstrated that LysK1-Sub5 accelerated wound healing and effectively reduced K. pneumoniae infection. This indicates LysK1-Sub5's potential as a topical antimicrobial agent against K. pneumoniae infections.