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Biomedical Journal
Volume 29, Issue 6 (11-2025)
IBJ 2025, 29(6): 384-396 |
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Noori A, Amini-Bayat Z, Mirdamadi S, Azizmohseni F, Arab S S, Moshref-Javadi M. Engineering a Potent Anti-MRSA Agent: The Development and Characterization of Chimeric Endolysin ZAM-MSC. IBJ 2025; 29 (6) :384-396
URL: http://ibj.pasteur.ac.ir/article-1-5200-en.html
URL: http://ibj.pasteur.ac.ir/article-1-5200-en.html
Atefeh Noori 
, Zahra Amini-Bayat * , Saeed Mirdamadi , Farzaneh Azizmohseni , Seyed Shahriar Arab , Mahtab Moshref-Javadi
, Zahra Amini-Bayat * , Saeed Mirdamadi , Farzaneh Azizmohseni , Seyed Shahriar Arab , Mahtab Moshref-Javadi
Abstract:
Background: 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.
Methods: 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.
Results: 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.
Conclusion: Our findings establish ZAM-MSC as a highly promising therapeutic candidate, combining computational precision with robust experimental validation.
Methods: 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.
Results: 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.
Conclusion: Our findings establish ZAM-MSC as a highly promising therapeutic candidate, combining computational precision with robust experimental validation.
Type of Study: Full Length/Original Article |
Subject:
Enzymology and Protein Chemistry
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