A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria
Background: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexit...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-02-01
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| Series: | Antibiotics |
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| Online Access: | https://www.mdpi.com/2079-6382/9/2/92 |
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| author | Gerard Boix-Lemonche Maria Lekka Barbara Skerlavaj |
| author_facet | Gerard Boix-Lemonche Maria Lekka Barbara Skerlavaj |
| author_sort | Gerard Boix-Lemonche |
| collection | DOAJ |
| description | Background: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i>. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (diSC<sub>3</sub>(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. Results: The ion channel-forming gramicidin D induced a rapid increase of diSC<sub>3</sub>(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. Conclusions: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs. |
| first_indexed | 2024-12-20T09:52:32Z |
| format | Article |
| id | doaj.art-0565e43214354d51b802bfd817677204 |
| institution | Directory Open Access Journal |
| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-12-20T09:52:32Z |
| publishDate | 2020-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-0565e43214354d51b802bfd8176772042022-12-21T19:44:33ZengMDPI AGAntibiotics2079-63822020-02-01929210.3390/antibiotics9020092antibiotics9020092A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive BacteriaGerard Boix-Lemonche0Maria Lekka1Barbara Skerlavaj2Department of Medicine, University of Udine, Piazzale Kolbe, 4, 33100 Udine, ItalyPolytechnic Department of Engineering and Architecture, University of Udine, Via delle Scienze 206, 33100 Udine, ItalyDepartment of Medicine, University of Udine, Piazzale Kolbe, 4, 33100 Udine, ItalyBackground: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i>. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (diSC<sub>3</sub>(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. Results: The ion channel-forming gramicidin D induced a rapid increase of diSC<sub>3</sub>(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. Conclusions: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.https://www.mdpi.com/2079-6382/9/2/92membrane depolarizationmembrane permeabilization<i>staphylococcus</i> spp.antimicrobial peptidespotentiometric dyepropidium iodidefluorescence-based assaymicroplate readerfe-sem |
| spellingShingle | Gerard Boix-Lemonche Maria Lekka Barbara Skerlavaj A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria Antibiotics membrane depolarization membrane permeabilization <i>staphylococcus</i> spp. antimicrobial peptides potentiometric dye propidium iodide fluorescence-based assay microplate reader fe-sem |
| title | A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria |
| title_full | A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria |
| title_fullStr | A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria |
| title_full_unstemmed | A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria |
| title_short | A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria |
| title_sort | rapid fluorescence based microplate assay to investigate the interaction of membrane active antimicrobial peptides with whole gram positive bacteria |
| topic | membrane depolarization membrane permeabilization <i>staphylococcus</i> spp. antimicrobial peptides potentiometric dye propidium iodide fluorescence-based assay microplate reader fe-sem |
| url | https://www.mdpi.com/2079-6382/9/2/92 |
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