Characterization of Three Different Endolysins Effective against Gram-Negative Bacteria

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacterial infections caused by Gram-negative pathogens pose a significant threat due to increasing antibiotic resistance.
• Bacteriophage-derived endolysins are enzymes with potent lytic activity against bacteria, offering a potential alternative to antibiotics.

Purpose of the Study:

• To identify, clone, and characterize endolysins from Escherichia coli bacteriophages.
• To evaluate the antibacterial activity and stability of these endolysins.
• To assess the in vivo efficacy of endolysins in a Galleria melonella infection model.

Main Methods:

• Gene identification and cloning from three Escherichia coli bacteriophages.
• Expression and purification of hexahistidine-tagged endolysins.
• Antibacterial activity assays against Gram-negative bacteria (e.g., E. coli, P. aeruginosa).
• Determination of minimum inhibitory concentrations (MIC).
• Enzymatic activity and stability assays (pH, temperature).
• In vivo efficacy testing in Galleria melonella larvae.

Main Results:

• Three endolysin genes were successfully cloned and expressed.
• Purified endolysins demonstrated broad-spectrum antibacterial activity against Gram-negative bacteria.
• Antibacterial activity was enhanced by N-terminal fusion with cecropin A, with MICs as low as 4 μg/mL.
• Endolysins remained enzymatically active across a pH range of 5-10 and were stable from 4-65 °C.
• In vivo studies in Galleria melonella confirmed the therapeutic potential of the endolysins.

Conclusions:

• Escherichia coli bacteriophage endolysins possess significant antibacterial properties against clinically relevant Gram-negative pathogens.
• Fusion with antimicrobial peptides like cecropin A can potentiate endolysin activity.
• The stability and demonstrated in vivo efficacy highlight the potential of these engineered endolysins as novel antimicrobial agents.