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Published on: December 19, 2020
Water-membrane partition and the mutant selection window of antimicrobial peptides: insights from liposome studies
Federico Carneri1, Cassandra Troiano2, Giuseppe Giaquinto2
1Department of Chemical Science and Technologies, Tor Vergata University of Rome, 00133 Rome, Italy; Photoinduced Processes and Technologies Doctoral School, Department of Chemistry, Biology and Biotechnology, Perugia University, 06123 Perugia, Italy.
Abstract:
The mutant selection window (MSW) is a range of antimicrobial concentrations, where some bacteria are killed, while others survive. Within this interval resistance may develop. Antimicrobial peptides (AMPs) are a promising class of antimicrobials that generally act by perturbing the integrity of bacterial membranes. Their MSW is typically narrower than that of traditional antibiotics, but it still encompasses about one order of magnitude of peptide concentrations. Phenotypic or genetic differences between individual cells may cause this heterogeneous bacterial response to AMPs. Therefore, we minimized the system complexity by investigating pore formation in liposomes with homogeneous size and composition. Surprisingly, the AMPs novicidin, P9-4, and Sub3 formed pores only in a fraction of vesicles, over a wide range of total peptide concentrations. By characterizing the water/membrane partition equilibrium of these three AMPs, we were able to report the vesicle-perturbing activity as a function of the membrane-bound peptide concentration. In this case, the curves became essentially step functions with well-defined (bound) concentration thresholds at which pores were formed in all liposomes. Therefore, the apparent heterogeneous effects of AMPs on vesicles were actually determined by variations in the fraction of membrane-bound peptides under different conditions, due to water-membrane partition. Unexpectedly, the thresholds coincided for all peptides in terms of bound amino acids per lipid (∼0.4), suggesting that the mechanism of pore formation primarily depends on the surface coverage by the AMPs.
Insights
Antimicrobial peptides (AMPs) can kill bacteria but also drive resistance. This study reveals AMP pore formation in liposomes depends on membrane-bound peptide concentration, not total concentration, with a universal threshold for pore formation.
Area of Science:
- Biochemistry
- Microbiology
- Membrane Biophysics
Background:
- The mutant selection window (MSW) describes antimicrobial concentrations where resistance can emerge.
- Antimicrobial peptides (AMPs) are potent antimicrobials targeting bacterial membranes, but their MSW is not fully understood.
- Heterogeneous bacterial responses to AMPs may stem from phenotypic or genetic variations.
Purpose of the Study:
- To investigate the mechanism of pore formation by AMPs in homogeneous liposomes.
- To determine if AMP activity is better described by total or membrane-bound peptide concentration.
- To identify potential thresholds for AMP-induced pore formation.
Main Methods:
- Studied pore formation in homogeneous liposomes using three AMPs: novicidin, P9-4, and Sub3.
- Characterized the water/membrane partition equilibrium of the AMPs.
- Quantified vesicle-perturbing activity as a function of membrane-bound peptide concentration.
Main Results:
- AMPs unexpectedly formed pores in only a fraction of liposomes across a wide concentration range.
- Vesicle perturbation followed step-function behavior when activity was plotted against membrane-bound peptide concentration.
- A consistent threshold of approximately 0.4 bound amino acids per lipid was observed for pore formation across all tested AMPs.
Conclusions:
- The apparent heterogeneity in AMP effects on liposomes is driven by variations in membrane-bound peptide concentration due to partitioning.
- Pore formation by these AMPs is primarily governed by surface coverage, indicated by a conserved threshold.
- This finding offers a unified mechanistic understanding of AMP pore formation and has implications for antimicrobial drug development.
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