Bacteriophage-derived endolysin EL07: antibacterial activity against multidrug-resistant Acinetobacter baumannii and its substrate recognition mechanism

Abstract Multidrug-resistant Acinetobacter baumannii has become a major threat to global public health, highlighting the need for alternative antimicrobial strategies. In this study, a novel lysin, EL07, derived from A. baumannii phage P4, was cloned, recombinantly expressed, and functionally characterized. Following outer membrane permeabilization, EL07 exhibited lytic activity against all 64 multidrug-resistant A. baumannii isolates tested and displayed cross-species activity against selected Pseudomonas aeruginosa and Escherichia coli strains. Kinetic analyses demonstrated rapid bacteriolytic activity, while fluorescence microscopy confirmed extensive membrane damage and bacterial inactivation after treatment. EL07 retained more than 80% of its activity over a pH range of 4.0–10.0 and maintained over 50% residual activity after exposure to 65 °C, indicating substantial physicochemical stability. To investigate the molecular basis of substrate recognition, interactions between EL07 and a peptidoglycan model (MGMG) were analyzed using density functional theory calculations, molecular docking, and 1-μs all-atom molecular dynamics simulations. Electrostatic potential analyses revealed complementary charge distributions between EL07 and MGMG. Molecular dynamics simulations showed stable complex formation throughout the trajectory, with a flexible loop region (residues 71–81) exhibiting pronounced conformational fluctuations near the substrate-binding interface. MM/GBSA calculations identified electrostatic interactions as the major favorable energetic contribution to binding and highlighted Glu55, Tyr76, and Leu77 as key residues involved in substrate recognition and stabilization. Collectively, these findings demonstrate that EL07 possesses broad antibacterial activity and robust environmental stability, while providing mechanistic insights into substrate recognition and binding, supporting its further development and rational engineering as an antibacterial protein targeting multidrug-resistant Gram-negative pathogens.