Abstract
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 Staphylococcus aureus and Staphylococcus epidermidis. 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 (diSC3(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 diSC3(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.
Highlights
The World Health Organization (WHO) identified antibiotic-resistant bacteria as one of the greatest threats to human health in the future [1]
The cytoplasmic membrane of bacteria may be regarded as a valid target for at least two reasons: (i) it is essential because it is the site where processes crucial for bacterial survival take place, and (ii) it is a conserved non-protein structure which cannot be modified without the risk of losing its functional and structural integrity, making the emergence of resistance less likely [5]
Knowing the complexity of living microbial membranes, there is a need for a deeper characterization of antimicrobial peptides (AMPs)-induced effects on membranes of live bacteria
Summary
The World Health Organization (WHO) identified antibiotic-resistant bacteria as one of the greatest threats to human health in the future [1]. Several antimicrobial peptides (AMPs) have been proposed as potential candidates for the development of novel antimicrobials [2,3,4] Among them, those exerting their bactericidal action by membrane permeabilization are interesting [2,4]. Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,30 -dipropylthiadicarbocyanine iodide (diSC3 (5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. 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
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