Small angle X-ray scattering as a high-throughput method to classify antimicrobial modes of action

Area of Science:

• Microbiology and Drug Discovery
• Structural Biology
• Biophysics

Background:

• Multi-drug resistant bacteria pose a global health threat, necessitating novel antimicrobial agents.
• Current methods for identifying antimicrobial modes of action are slow, costly, and not high-throughput.
• Antimicrobial peptides are promising candidates, but understanding their mechanism is crucial for development.

Purpose of the Study:

• To explore small angle X-ray scattering (SAXS) as a high-throughput method for classifying antimicrobial modes of action.
• To rapidly assess if novel antimicrobial peptides have distinct mechanisms compared to existing antibiotics.
• To support efficient decision-making in antimicrobial drug development.

Main Methods:

• Utilized SAXS to analyze ultrastructural changes in Escherichia coli cells treated with a novel antimicrobial peptide and characterized antibiotics.
• Employed principal component analysis (PCA) to analyze SAXS data and correlated results with Transmission Electron Microscopy (TEM) images.
• Measured nanoscale information averaged over a large cell population (approx. one million cells) in seconds.

Main Results:

• SAXS effectively provided rapid feedback on drug-induced ultrastructural alterations in E. coli cells.
• Ultrastructural changes were found to be dependent on the specific antibiotic and its mode of action.
• The novel antimicrobial peptide exhibited a distinct mode of action compared to all tested antibiotics, including polymyxin B.

Conclusions:

• SAXS is a valuable, structure-sensitive tool for rapid mode of action classification in drug discovery.
• The high-throughput nature of SAXS accelerates the screening and development of novel antimicrobial candidates.
• SAXS can differentiate novel antimicrobial peptides with unique mechanisms, crucial for combating multi-drug resistant bacteria.