Expression, purification and broad-spectrum antibacterial characterization of Pseudomonas aeruginosa phage PPAT endolysin

The prevalence of multidrug-resistant Gram-negative bacteria poses a serious threat to clinical anti-infective therapy, and bacteriophage-encoded endolysins have emerged as promising alternatives to conventional antibiotics due to their high efficiency and low risk of resistance development. In this study, the endolysin gene lysPPAT from Pseudomonas aeruginosa bacteriophage PPAT was cloned and heterologously expressed in Escherichia coli , and the recombinant protein was successfully purified. Bioinformatic analysis revealed that LysPPAT contains a conserved phage lysozyme domain without transmembrane regions or signal peptides, consistent with typical endolysin characteristics. In vitro assays demonstrated that LysPPAT exhibits broad-spectrum antibacterial activity against a subset of tested Gram-negative and Gram-positive pathogens, including clinically resistant strains of P. aeruginosa , Klebsiella pneumoniae , Acinetobacter baumannii , Escherichia coli , and Staphylococcus aureus , even in the absence of outer membrane permeabilizers. The optimal enzymatic activity was observed at 37 °C and pH 8.0. Scanning electron microscopy and peptidoglycan degradation assays confirmed that LysPPAT directly disrupts bacterial cell walls, with degradation activity against P. aeruginosa peptidoglycan comparable to that of lysozyme. Molecular docking revealed stable hydrogen bond interactions between LysPPAT and peptidoglycan, with a docking score of −4.56 kcal·mmol −1 . Amphipathic α-helices were predicted to be the structural basis for outer membrane penetration. Additionally, LysPPAT significantly reduced mature P. aeruginosa and S. aureus biofilms biomass. Collectively, LysPPAT represents a novel broad-spectrum endolysin with great potential for combating multidrug-resistant bacterial infections.