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Updated: Jun 6, 2025
Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings
Published on: August 19, 2021
Evaluating the antibacterial activity of engineered phage ФEcSw endolysin against multidrug-resistant Escherichia
Maheswaran Easwaran1, Rajiv Gandhi Govindaraj2, Misagh Naderi3
1Department of Research Analytics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India.
Objective:
The emergence of bacteriophage-encoded endolysins hold significant promise as novel antibacterial agents, particularly against the growing threat of antibiotic-resistant bacteria. Therefore, we investigated the phage ФEcSw endolysin to enhance the lytic activity against multi-drug-resistant Escherichia coli Sw1 through site-directed mutagenesis (SDM) guided by in silico identification of critical residues.
Methods:
A computational analysis was conducted to elucidate the protein folding pattern, identify the active domains, and recognize critical residues of ФEcSw endolysin. Structural similarity-based docking simulations were employed to identify residues potentially involved in both recognition and cleavage of the bacterial peptidoglycan. Phage endolysin was amplified, cloned, expressed, and purified from phage ФEcSw. Pure endolysin (EL) activity was subsequently validated through SDM.
Results:
Our studies revealed both open and closed conformations of ФEcSw endolysin within specific residue ranges (51-60 and 128-141). Notably, the active site was identified and contains the crucial catalytic residues, Glu19 and Asp34. A time-kill assay demonstrated that the holin (HL) - EL effectively reduced E. coli Sw1 growth by 46% within 12 h. Furthermore, treatment with HL, EL, and HL-EL significantly increased bacterial membrane permeability (11%, 74%, and 85%, respectively) within just 1 h. Importantly, SDM identified a double mutant (K19/H34) of the endolysin exhibiting the highest lytic activity compared to the wild-type and other mutants (E19D, E19K, D34E, and D34H) due to increase net charge from +3.23 to +6.29.
Conclusions:
Our findings demonstrate that phage endolysins and HLs or engineered endolysin hold significant potential as therapeutic agents to combat multidrug-resistant bacterial infections.
