Elsevier

Peptides

Volume 31, Issue 10, October 2010, Pages 1811-1820
Peptides

Interaction of cationic antimicrobial peptides with phospholipid vesicles and their antibacterial activity

https://doi.org/10.1016/j.peptides.2010.06.021Get rights and content

Abstract

We have designed and synthesized a series of cationic α-helical AMPs with improved antibacterial activity and selectivity against a broad spectrum of G(+) and G(−) bacteria. In the current study, we intended to gain further insight into the mechanisms of action between AMPs and cellular membranes using model liposomes of various phospholipid compositions. Circular dichroism measurements showed that AMPs adopted amphipathic α-helical conformation in the presence of negatively charged vesicles (DOPC/DOPG = 1:3), while they were largely unstructured when incubated with neutral vesicles (DOPC). The interaction of AMPs with phospholipid vesicles were further analyzed by calcein leakage experiments. AMPs exhibited weak dye-leakage activity for DOPC (neutral) vesicles, while they effectively induced calcein leakage when interacted with DOPC/DOPG-entrapped vesicles. These results indicated that our newly designed cationic AMPs did show preferences for bacteria-mimicking anionic membranes. All of them exert their cytolytic activity by folding into an amphipathic helix upon selectively binding and insertion into the target membrane, leading to breakdown of the membrane structure, thus causing leakage of cell contents, resulting finally in cell death. Elucidating the mechanism of the membranolytic activity of AMPs may facilitate the development of more effective antimicrobial agents.

Research highlights

▶ Cationic AMPs adopt amphipathic α-helical conformation in the presence of negatively charged vesicles (DOPC/DOPG = 1:3). ▶ Cationic AMPs effectively induce calcein-leakage when interacted with DOPC/DOPG-entrapped vesicles. ▶ Cationic AMPs show preferences for bacteria-mimicking anionic membranes. ▶ Cationic AMPs exert their cytolytic activity by folding into an amphipathic helix upon selectively binding and insertion into the target membrane, leading to breakdown of the membrane structure, thus causing leakage of cell contents, resulting finally in cell death. ▶ Elucidating the mechanism of the membranolytic activity of cationic AMPs may facilitate the development of more effective antimicrobial agents.

Introduction

The abuse of traditional antibiotics has led to the growing emergence of many drug-resistant bacterial strains [23]. In recent years, cationic antimicrobial peptides (AMPs) have drawn much attention as a promising antibacterial agent that could overcome the resistance problem [11], [12], [14]. This is due to their ability to disrupt bacterial membranes via non-specific electrostatic interactions with the membrane lipid components. There are two common and important criteria for functionally active AMPs; a net cationic charge and the ability to adopt an amphipathic structure, where the hydrophobic and hydrophilic parts are oriented in opposite phases upon interaction with negatively charged bacterial membranes.

The possible mechanisms of action of AMPs fall into two categories: (1) the formation of pores in bacterial membranes through either transmembrane structures (barrel stave and toroidal pore models) or (2) by dissolving the membrane in a detergent-like manner (carpet model) [1], [2], [3], [24], [27]. Then they may subsequently lead to breakdown of the transmembranal potential, thus cause leakage of cell contents, resulting finally in cell death [10]. An AMP that works solely by one of the above mechanisms is not common and the actually observed mechanism usually depends on the lipid composition as well as on the lipid/peptide ratio.

A critical feature of the cationic AMPs is their ability to selectively distinguish bacterial from mammalian cells based on the fact that the lipid composition of their membranes is different. The outer leaflet of human erythrocytes, typically for eukaryotic cell membranes, is composed of neutral lipid components, mainly PC, PE, sphingomyelin and cholesterol. In contrast, the inner membrane of Escherichia coli, as a model for bacterial membranes, contains mainly PE together with substantial amounts of negatively charged phospholipids, namely PG and cardiolipin [9], [17], [19], [21].

In our previous studies, we have reported the design and synthesis of a series of cationic α-helical peptides of 20 a.a. based on four structural parameters, including charge, polar angle, hydrophobicity, and hydrophobic moment [7]. Four of the synthetic AMPs were found to have enhanced antimicrobial activity, decreased hemolytic activity, and improved selectivity over the two natural potent AMPs, magainin 2a and pleurocidin while maintaining broad spectrum activity for Gram-negative and Gram-positive bacteria. In the current study, our main goals were to investigate the selectivity of the peptides, which have the same helical structure, for phospholipid vesicles composed of different ratios of DOPC and DOPG. We believe that by elucidating the mechanism of the membranolytic activity of AMPs may facilitate the development of more effective antimicrobial agents.

Section snippets

Bacterial strains

Two Gram-positive (Staphylococcus aureus, BCRC 10780 and Listeria monocytogenes, BCRC 14845) and three Gram-negative bacteria (E. coli, BCRC 10675 and Pseudomonas aeruginosa, BCRC 10944), including one important marine pathogen (Vibrio parahaemolyticus) kindly provided by Dr. Tsun-Yung Kuo's lab as clinical isolate from diseased aquaculture, were selected for measurement of the antibacterial activity of AMPs used in the current study. S. aureus was cultured in tryptic soy broth (TSB) at 37 °C,

AMP design and synthesis

The AMPs we are using in the current study were designed and synthesized in our previous work according to several structural parameters, including charge, polar angle, hydrophobicity, and hydrophobic moment [7]. The AMPs were estimated from their primary sequence to be amphipathic when they adopt α-helical structures. The hydrophobicity and helicity of cationic AMPs are important for their antibacterial activity [8]. According to our design strategy, the AMPs used in this study should assume

Discussion

In our previous studies, we have reported the design and synthesis of a series of cationic α-helical peptides of 20 a.a. based on four structural parameters, including charge, polar angle, hydrophobicity, and hydrophobic moment [7]. Four of the synthetic AMPs, Q4, Q6, H1 and M1 were found to have enhanced antimicrobial activity, decreased hemolytic activity, and improved selectivity over the two natural potent AMPs, magainin 2a and pleurocidin while maintaining broad spectrum activity for

Acknowledgments

This work was supported in part by grants NSC 94-2317-B-197-001 and NSC 95-2311-B-197-003 from the National Science Council, Taiwan, Republic of China. We would like to thank Mr. Jung-Chun Chiang, Ms. Min-Ju Pei, Wei-Ter Yang and Hui-Chun Yu for their excellent technical assistance.

References (28)

  • V.E. Vaskovsky et al.

    A universal reagent for phospholipid analysis

    J Chromatogr

    (1975)
  • K.A. Brogden

    Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?

    Nat Rev Microbiol

    (2005)
  • W.J. Chen et al.

    Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding

    Biochemistry

    (2004)
  • W.J. Chen et al.

    Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

    Protein Sci

    (2004)
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