Design, Characterization, and Efficacy of ASEnd: A Novel Chimeric Endolysin for Targeting Methicillin-Resistant...

The emergence of antibiotic-resistant pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), represents a major public health threat, with limited treatment options. Bacteriophage-derived endolysins and their engineered chimeric derivatives have attracted attention as alternative antibacterial agents due to their rapid bacteriolytic activity, target specificity, and low resistance propensity. ASEnd was constructed by fusing the enzymatically active LYZ2 domain from S. aureus phage φMR11 with the SH3b-type cell wall-binding domain from phage K via a flexible (G4S)₃ linker. Structural integrity was assessed in silico using Ramachandran plot analysis, ERRAT, Verify3D, and ProSA-web. The recombinant protein was expressed in Escherichia coli BL21 (DE3) and purified by immobilized metal affinity chromatography. Antibacterial activity was evaluated against MRSA, methicillin-susceptible S. aureus (MSSA), and other bacterial strains using disk diffusion, turbidity reduction, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. pH tolerance, storage stability, and cytotoxicity toward HEK-293 cells were also examined. ASEnd was obtained as a soluble 34.7 kDa protein with favorable structural metrics (>99% of residues in favored Ramachandran regions). The protein exhibited potent activity against S. aureus, producing inhibition zones up to 19 mm and rapidly reducing turbidity at low concentrations. ASEnd demonstrated MIC and MBC values of 1.56 and 3.125 µg/mL, respectively, representing a 16-fold improvement over LYZ2. The enzyme retained activity between pH 6 and 8, but declined significantly below pH 5, and gradual loss was observed during storage at 4°C (21%, 42%, and 78% reductions after 1, 7, and 14 days), with complete inactivation after 3 weeks. ASEnd showed no cytotoxicity toward HEK-293 cells and displayed selective activity against MRSA and MSSA, with limited cross-reactivity against Streptococcus pyogenes (2.4-fold lower) and no effect on E. coli. These findings identify ASEnd as a rationally designed chimeric endolysin with enhanced potency and selectivity against S. aureus and a favorable safety profile. Further stability optimization, clinical isolate testing, and in vivo validation are required to establish its therapeutic potential.