Engineering a Potent Anti-MRSA Agent: The Development and Characterization of Chimeric Endolysin ZAM-MSC

The increasing prevalence of S. aureus, especially methicillin-resistant strains, poses a major healthcare threat due to limited therapies. To address this challenge, we engineered the chimeric endolysin ZAM-MSC as a potent antibiotic alternative, using domain-fusion strategies to enhance antibacterial activity. We designed ZAM-MSC by integrating the catalytic (M23) and cell wall-binding (SH3b) domains of lysostaphin with the catalytic domain (CHAP) from endolysin SAL-1. Structural optimization was performed using AlphaFold2 prediction, AutoDock Vina docking, and GROMACS simulations to evaluate domain interactions, protein stability, and binding dynamics.

The chimeric construct was cloned into pCold I, expressed in E. coli, and purified under solubility-optimized conditions. Purified ZAM-MSC, at a minimum concentration of 3 μg, reduced bacterial optical density within 15 minutes, demonstrating potent lytic activity. Thermal stability assays indicated that ZAM-MSC retained its enzymatic activity over 80-90% across 4-37 °C, with only a 10-20% decrease at 25-37 °C after 30 minutes. NaCl stability tests revealed maximal activity in the absence of NaCl, with gradual reduction in enzyme activity by increasing NaCl concentrations.

Cytotoxicity analysis via MTT assay on L929 fibroblast cells showed cell viabilities of ~85-90% ± 5% at the highest enzyme concentrations tested, with no detectable cytotoxic effect compared to untreated controls. Hemolysis assays confirmed nearly 100% red blood cell integrity across all tested enzyme concentrations, supporting its biocompatibility with mammalian cells.

Our findings establish ZAM-MSC as a highly promising therapeutic candidate, combining computational precision with robust experimental validation.