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A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

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.

SUBMITTER: Boix-Lemonche G

PROVIDER: S-EPMC7168298 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

Boix-Lemonche Gerard G Lekka Maria M Skerlavaj Barbara B

Antibiotics (Basel, Switzerland) 20200219 2

<h4>Background</h4>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 ep ...[more]

PMID: 32093104

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Project description:Antimicrobial peptides (AMPs) are ubiquitous amongst living organisms and are part of the innate immune system with the ability to kill pathogens directly or indirectly by modulating the immune system. AMPs have potential as a novel therapeutic against bacteria due to their quick-acting mechanism of action that prevents bacteria from developing resistance. Additionally, there is a dire need for therapeutics with activity specifically against Gram-negative bacterial infections that are intrinsically difficult to treat, with or without acquired drug resistance. Development of new antibiotics has slowed in recent years and novel therapeutics (like AMPs) with a focus against Gram-negative bacteria are needed. We designed eight novel AMPs, termed PHNX peptides, using ab initio computational design (database filtering technology combined with the novel positional analysis on APD3 dataset of AMPs with activity against Gram-negative bacteria) and assessed their theoretical function using published machine learning algorithms, and finally, validated their activity in our laboratory. These AMPs were tested to establish their minimum inhibitory concentration (MIC) and half-maximal effective concentration (EC50) under CLSI methodology against antibiotic resistant and antibiotic susceptible Escherichia coli and Staphylococcus aureus. Laboratory-based experimental results were compared to computationally predicted activities for each of the peptides to ascertain the accuracy of the computational tools used. PHNX-1 demonstrated antibacterial activity (under high and low-salt conditions) against antibiotic resistant and susceptible strains of Gram-positive and Gram-negative bacteria and PHNX-4 to -8 demonstrated low-salt antibacterial activity only. The AMPs were then evaluated for cytotoxicity using hemolysis against human red blood cells and demonstrated some hemolysis which needs to be further evaluated. In this study, we successfully developed a design methodology to create synthetic AMPs with a narrow spectrum of activity where the PHNX AMPs demonstrated higher antibacterial activity against Gram-negative bacteria compared to Gram-positive bacteria. Thus, these peptides present novel synthetic peptides with a potential for therapeutic use. Based on our findings, we propose upfront selection of the peptide dataset for analysis, an additional step of positional analysis to add to the ab initio database filtering technology (DFT) method, and we present laboratory data on the novel, synthetically designed AMPs to validate the results of the computational approach. We aim to conduct future in vivo studies which could establish these AMPs for clinical use.

Project description:The activity of antimicrobial peptides (AMPs) has been investigated extensively using model membranes composed of phospholipids or lipopolysaccharides in aqueous environments. However, from a biophysical perspective, there is a large scientific interest regarding the direct interaction of membrane-active peptides with whole bacteria. Working with living bacteria limits the usability of experimental setups and the interpretation of the resulting data because of safety risks and the overlap of active and passive effects induced by AMPs. We killed or inactivated metabolic-active bacteria using ?-irradiation or sodium azide, respectively. Microscopy, flow cytometry, and SYTOX green assays showed that the cell envelope remained intact to a high degree at the minimal bactericidal dose. Furthermore, the tumor-necrosis-factor-?-inducing activity of the lipopolysaccharides and the chemical lipid composition was unchanged. Determining the binding capacity of AMPs to the bacterial cell envelope by calorimetry is difficult because of an overlapping of the binding heat and metabolic activities of the bacteria-induced by the AMPs. The inactivation of all active processes helps to decipher the complex thermodynamic information. From the isothermal titration calorimetry (ITC) results, we propose that the bacterial membrane potential (??) is possibly an underestimated modulator of the AMP activity. The negative surface charge of the outer leaflet of the outer membrane of Gram-negative bacteria is already neutralized by peptide concentrations below the minimal inhibitory concentration. This proves that peptide aggregation on the bacterial membrane surface plays a decisive role in the degree of antimicrobial activity. This will not only enable many biophysical approaches for the investigation between bacteria and membrane-active peptides in the future but will also make it possible to compare biophysical parameters of active and inactive bacteria. This opens up new possibilities to better understand the active and passive interaction processes between AMPs and bacteria.

Dataset Information

A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

Publications

A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

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