Overcoming outer membrane barrier in gram-negative bacteria by PCNP-PVP-SE1 phage endolysin: Evidence from molecular docking, molecular dynamics and experimental analyses.

Abstract

The increasing prevalence of antimicrobial resistance among Gram-negative pathogens necessitates the development of alternative antibacterial strategies. Bacteriophage-derived endolysins represent a promising class of antimicrobials; however, their limited ability to traverse the outer membrane of Gram-negative bacteria restricts their exogenous application. In this study, the native PVP-SE1 endolysin and a PCNP-fused Artilysin variant were expressed, purified, and comprehensively characterized using in vitro and in silico approaches. Antibacterial activity was evaluated against Escherichia coli and Salmonella Typhimurium using disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays, each performed in biological triplicate (n = 3). While the native endolysin required EDTA to exert antibacterial activity, the engineered variant was independently bactericidal, producing significantly larger inhibition zones and a significantly more rapid reduction in viable bacterial counts (p < 0.05). Structural modeling and molecular docking confirmed preservation of the catalytic core and substrate-binding residues following N-terminal PCNP fusion. Molecular dynamics simulations and MM-PBSA binding free-energy analyses indicated that the engineered complex achieved a more favorable binding free energy than the native form, driven primarily by stronger van der Waals and electrostatic interactions rather than hydrogen bonding. Collectively, these findings indicate that N-terminal PCNP fusion confers statistically supported bactericidal activity against E. coli and Salmonella Typhimurium with a markedly reduced dependence on EDTA, without compromising catalytic integrity, supporting further evaluation of PCNP-fused Artilysins as antimicrobial candidates.