Development of Chimera AMP-Endolysin with Wider Spectra Against Gram-Negative Bacteria Using High-Throughput Assay

Area of Science:

• Microbiology
• Biotechnology
• Drug Discovery

Background:

• Bacteriophage-derived endolysins show promise as alternatives to conventional antimicrobials.
• Developing effective endolysins against Gram-negative bacteria is challenging due to the need for outer membrane penetration.
• Existing antimicrobial strategies face increasing resistance, necessitating novel therapeutic agents.

Purpose of the Study:

• To establish an efficient endolysin development pipeline for Gram-negative bacteria.
• To identify novel natural endolysins effective against Acinetobacter baumannii.
• To engineer chimeric antimicrobial peptide-endolysins (AMP-endolysins) with enhanced efficacy and broader spectra.

Main Methods:

• In silico analysis of 273 bacterial genomes to identify potential endolysin genes.
• High-throughput screening of 192 recombinant endolysins for peptidoglycan degradation activity.
• Construction and screening of hundreds of chimeric AMP-endolysins against Gram-negative pathogens.
• Determination of minimum inhibitory concentrations (MICs) and antimicrobial spectra.

Main Results:

• Identified natural endolysins capable of degrading Acinetobacter baumannii peptidoglycan.
• Developed four chimeric AMP-endolysins against A. baumannii with MICs of 4-8 μg/mL.
• Two chimeric endolysins demonstrated efficacy against Pseudomonas aeruginosa and Escherichia coli at concentrations below 32 μg/mL.

Conclusions:

• The combined in silico and high-throughput screening approach is efficient for discovering novel endolysins.
• Engineered AMP-endolysins represent a promising strategy for combating Gram-negative bacterial infections.
• This methodology can be adapted for developing endolysins against other Gram-negative bacterial targets.