Discovery of antimicrobials by massively parallelized growth assays (Mex)

Philipp Koch,Niko Beerenwinkel,Martin Held,Steven Schmitt,Sven Panke,Mathias Cardner

doi:10.1038/s41598-022-07755-7

Philipp Koch, Niko Beerenwinkel + Show 4 more

Open Access

https://doi.org/10.1038/s41598-022-07755-7

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Abstract

The number of newly approved antimicrobial compounds has been steadily decreasing over the past 50 years emphasizing the need for novel antimicrobial substances. Here we present Mex, a method for the high-throughput discovery of novel antimicrobials, that relies on E. coli self-screening to determine the bioactivity of more than ten thousand naturally occurring peptides. Analysis of thousands of E. coli growth curves using next-generation sequencing enables the identification of more than 1000 previously unknown antimicrobial peptides. Additionally, by incorporating the kinetics of growth inhibition, a first indication of the mode of action is obtained, which has implications for the ultimate usefulness of the peptides in question. The most promising peptides of the screen are chemically synthesized and their activity is determined in standardized susceptibility assays. Ten out of 15 investigated peptides efficiently eradicate bacteria at a minimal inhibitory concentration in the lower µm or upper nm range. This work represents a step-change in the high-throughput discovery of functionally diverse antimicrobials.

Highlights

The number of newly approved antimicrobial compounds has been steadily decreasing over the past 50 years emphasizing the need for novel antimicrobial substances
For technical reasons (Methods), we applied a cut-off of 42 amino acids in peptide chain length and selected Similars with at least 62.2% sequence
Similar HFIAP-14545 NCBI inhibited growth of these strains (MICs: 0.4–5.6 μm; Table S2), which suggests a broad activity spectrum even though Mex screening was performed in another host. These results indicated that we screened the peptide library synthesized cytosolically, Mex-active peptides strongly inhibited growth when added to cells externally and that the employed E. coli lab strain was suited well for the identification of peptides active against other bacterial species

Summary

Introduction

The number of newly approved antimicrobial compounds has been steadily decreasing over the past 50 years emphasizing the need for novel antimicrobial substances. We present Mex, a method for the high-throughput discovery of novel antimicrobials, that relies on E. coli self-screening to determine the bioactivity of more than ten thousand naturally occurring peptides. Natural compounds are fundamental for drug discovery as they provide the biological relevance and structural diversity required to identify drug-like p harmacophores[1]. Owing to their high structural complexity and their ability to penetrate tissues and membranes, peptides are becoming increasingly important for many therapeutic areas[2]. Peptides are expressed from their encoding DNA template and accumulate either in the cytosol, the periplasm or at the bacterial s urface[6] If antimicrobial, their expression negatively impacts the proliferation rate or survival of the expressing cell. The method delivered a rich collection of functionally diverse and highly active antimicrobial peptides

Methods

Results

Conclusion