Macromolecule Biosynthesis Assay and Fluorescence Spectroscopy Methods to Explore Antimicrobial Peptide Mode(s) of Action
Antimicrobial peptides (AMPs) are viable alternatives to the currently available antimicrobials, and numerous studies have investigated their possible use as therapeutic agents for specific clinical applications. AMPs are a diverse class of antimicrobials that often act upon the bacterial cell membrane but may exhibit additional modes of action. Identification of the multiple modes of action requires a comprehensive study at subinhibitory concentrations and careful data analysis since additional modes of action can be eclipsed by AMP action on the cell membrane.
Techniques that measure the biosynthesis rate of macromolecules (e.g., DNA, RNA, protein, and cell wall) and the cytoplasmic membrane proton motive force (PMF) energy can help to unravel the diverse modes of action of AMPs. Here, we present an overview of macromolecule biosynthesis rate measurement and fluorescence spectroscopy methods to identify AMP mode(s) of action. Detailed protocols designed to measure inhibition of DNA, RNA, protein, and cell wall synthesis or membrane de-energization are presented and discussed for optimal application of these two techniques as well as to enable accurate interpretation of the experimental findings.
Key wordsAntimicrobial peptide Mode(s) of action Macromolecule biosynthesis Fluorescence spectroscopy Proton motive force Membrane de-energization/depolarization
The work reported here has been supported by grants from the University of Copenhagen Centre for Control of Antimicrobial Resistance (UC-Care) and Zoetis.
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