Study on the Cloning, Expression, and Antimicrobial Activity of the Holin Gene from Klebsiella pneumoniae Bacteriophage
Phage holins can form pores in the cell membrane, allowing endolysins to enter the cell wall and degrade peptidoglycan, thereby releasing mature phage particles or leading to cell lysis and death. Both endolysins and holins demonstrate significant potential in controlling bacterial infections. This study investigates the holin protein encoded by Klebsiella pneumoniae phage vB_Kp_XP4. The results indicate that the HolKpV318 gene is 252 bp in length and encodes a protein consisting of 83 amino acid residues, belonging to the phage Holin family, with a predicted molecular weight of approximately 9 kDa. HolKpV318 exhibits concentration- and time-dependent antimicrobial effects, with a minimum inhibitory concentration (MIC) of about 4 µg/mL. When combined with HolKpV318, the MIC of Sulfamethazine decreases to 1/256 of its single-agent MIC. HolKpV318 demonstrates optimal anti-biofilm activity at a concentration of 12 µg/mL. Treatment with this protein significantly increases host bacterial cell membrane permeability, ultimately leading to cell lysis. HolKpV318 exhibits the highest antimicrobial activity at 50°C and effectively inhibits host bacterial growth within a pH range of 3–10. Furthermore, HolKpV318 exhibits lytic activity against various clinical pathogenic bacteria, including Klebsiella pneumoniae, Klebsiella pasteurii, Klebsiella grimontii, Enterobacter hormaechei, and Raoultella planticola. It demonstrates particularly good lytic ability against Klebsiella pneumoniae strains carrying carbapenemase genes (such as KPC-2, NDM-1, and NDM-5) and those of different ST and K types, including hypervirulent strains. These results indicate that the holin HolKpV318 exhibits strong broad-spectrum antibacterial activity, holding promise for the development of novel antibacterial agents.