Functional whole-colony screening method to identify antimicrobial peptides

Area of Science:

• Biotechnology
• Microbiology
• Drug Discovery

Background:

• Antimicrobial peptides (AMPs) are crucial in innate immunity.
• Discovering novel AMPs is essential to combat rising antibiotic resistance.
• Existing screening methods can be time-consuming and low-throughput.

Purpose of the Study:

• To develop and validate a high-throughput screening method for identifying antimicrobial peptides (AMPs).
• To assess the efficacy of a novel dye inclusion assay for detecting bacterial viability antagonism.
• To screen a synthetic oligonucleotide library for novel AMPs.

Main Methods:

• A high-throughput method using a vital dye inclusion assay was developed.
• Colonies were grown on a membrane and transferred to an inductive medium with vital dye.
• Compromised cell membranes due to peptide expression allowed dye infusion for visual identification.

Main Results:

• The method successfully identified three antimicrobial peptides (L1, L3, L8) from a library of 100,000 potential peptides.
• Peptide L1 exhibited concentration-dependent bactericidal effects against Gram-negative bacteria and biostatic activity against Gram-positive bacteria.
• Peptide L8 showed bacteriostatic and potentially bactericidal effects against multiple Gram-negative bacteria.

Conclusions:

• The developed high-throughput assay based on filter-bound colony arrays and vital dye inclusion is effective for AMP identification.
• This method accelerates the discovery of novel antimicrobial peptides.
• The identified peptides L1 and L8 demonstrate significant antimicrobial potential.