title: Macromolecule Biosynthesis Assay and Fluorescence Spectroscopy Methods to Explore Antimicrobial Peptide Mode(s) of Action.
abstract: 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.
authors.affiliation: Department of Biomedical Sciences, Ross University School of Veterinary Medicine, PO Box 334, Basseterre, West Indies, St Kitts and Nevis. bimal@sund.ku.dk.
authors.forename: Bimal
authors.initials: B
authors.lastname: Jana
authors.affiliation: Department of Biomedical Sciences, Ross University School of Veterinary Medicine, PO Box 334, Basseterre, West Indies, St Kitts and Nevis.
authors.forename: Kristin Renee
authors.initials: KR
authors.lastname: Baker
authors.affiliation: Department of Biomedical Sciences, Ross University School of Veterinary Medicine, PO Box 334, Basseterre, West Indies, St Kitts and Nevis.|Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Luca
authors.initials: L
authors.lastname: Guardabassi
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D046911:Macromolecular Substances
keywords: Antimicrobial peptide; Fluorescence spectroscopy; Macromolecule biosynthesis; Membrane de-energization/depolarization; Mode(s) of action; Proton motive force