title: High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides' activity on various yeast species.
abstract: New antifungal compounds that circumvent the resistance of the pathogen by directly damaging yeast cell surface structures are promising agents for the treatment of fungal infections, due to their different mechanism of action from current clinically used antifungal drugs. We present here a rapid and cost-effective fluorescence method suitable for identifying new potent drugs that directly target yeast cell surface structures, causing cell permeabilization and thus bypassing the multidrug resistance mechanisms of pathogens. The fluorescence assay enabled us to detect with high sensitivity damage to the Candida plasma membrane (its hyperpolarization and permeabilization) as a result of short-term exposure to the antifungal compounds. Results can be obtained in 1-2h with minimal effort and consumption of the tested compounds, also 96 samples can be analysed simultaneously. We used this method to study antimicrobial peptides isolated from the venom of bees and their synthetic analogs, compare the potency of the peptides and determine their minimal effective concentrations. The antimicrobial peptides were able to kill yeast cells at low concentrations within a 15-min treatment, the LL-III peptide exhibited a broad spectrum of antifungal activity on various Saccharomyces, pathogenic Candida and osmotolerant yeast species.
authors.affiliation: Department of Membrane Transport, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Průmyslová 595, 252 42, Vestec, Czech Republic. Electronic address: Marie.Kodedova@fgu.cas.cz.
authors.forename: Marie
authors.initials: M
authors.lastname: Kodedová
authors.affiliation: Department of Membrane Transport, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Průmyslová 595, 252 42, Vestec, Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Sychrová
mesh_terms: D000935:Antifungal Agents / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D005658:Fungi / Q000166:cytology; D057166:High-Throughput Screening Assays / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides
keywords: Antimicrobial peptide; Candida; Membrane potential; Microplate reader; diS-C(3)(3) assay