Interaction between Antibacterial Peptide Apep10 and Escherichia coli Membrane Lipids Evaluated Using Liposome as Pseudo-Stationary Phase

RESEARCH ARTICLE Interaction between Antibacterial Peptide Apep10 and Escherichia coli Membrane Lipids Evaluated Using Liposome as PseudoStationary Phase Wenting Tang*☯, Chuanfen Pu☯, Man Li School of Food Science and Engineering, Qingdao Agricultural University, Qingdao, 266109, China a11111 ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Tang W, Pu C, Li M (2017) Interaction between Antibacterial Peptide Apep10 and Escherichia coli Membrane Lipids Evaluated Using Liposome as Pseudo-Stationary Phase. PLoS ONE 12(1): e0164594. doi:10.1371/journal. pone.0164594 Editor: Andreas Hofmann, Griffith University, AUSTRALIA Received: July 19, 2016 Accepted: September 27, 2016 Published: January 4, 2017 Copyright: © 2017 Tang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Liposomes constructed from Escherichia coli membrane lipids were used as a pseudo-stationary phase in capillary electrophoresis and immobilised liposome chromatography to evaluate the interaction between antibacterial peptide (ABP) Apep10 and bacterial membrane lipids. The peptide mobility decreased as the concentration of liposomes increased, providing evidence for the existence of this interaction. The binding constant between Apep10 and the Escherichia coli membranes lipid liposome was higher than that of Apep10 with a mixed phospholipids liposome at the same temperature. The capillary electrophoresis results indicate that the binding ability of Apep10 with a liposome was dependent on the liposome’s lipid compositions. Thermodynamic analysis by immobilised liposome chromatography indicated that hydrophobic and electrostatic effects contributed to the partitioning of Apep10 in the membrane lipids. The liposomes constructed from bacterial membrane lipid were more suitable as the model membranes used to study dynamic ABP/membrane interactions than those constructed from specific ratios of particular phospholipids, with its more biomimetic phospholipid composition and contents. This study provides an appropriate model for the evaluation of ABP-membrane interactions. Data Availability Statement: All relevant data are within the paper. Introduction Funding: This study was supported by University Science and Technology Project of Shandong Province of China (no. J16LE22, http://www.sdedu. gov.cn/, WT), Doctoral Science Foundation of Shandong Province of China (no. BS2015SW019, http://www.sdstc.gov.cn/, WT), and Advanced Talents Foundation of Qingdao Agricultural University (no. 6631115030, www.qau.edu.cn, WT). The funders had no role in study design, data Antibacterial peptides (ABPs) have attracted much attention due to their potential to overcome bacterial resistance and are promising candidates for novel antibiotics [1], [2]. It is well established that the first stage of ABPs’ action is to combine with the bacterial cell membrane [3–5]. Therefore, knowledge of this interaction is vital to understand their antibacterial mechanism. However, the natural cell membrane has a very short lifespan, which makes it an imperfect candidate for research. To overcome this shortcoming, self-assembled vesicles known as liposomes have been widely used as model membranes, due to the structural similarities between liposomes and the natural cell membrane, both of which are phospholipid bilayers [6–9]. PLOS ONE | DOI:10.1371/journal.pone.0164594 January 4, 2017 1 / 10 Interaction between Apep10 and Membrane Lipids Evaluated Using Liposome collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Immobilised liposome chromatography (ILC), a technique in which liposomes are immobilised in stationary phases for liquid chromatography, has been studied as a membrane analysis technique [10–13]. However, the liposomes used for this method are usually constructed from specific ratios of particular phospholipids that cannot mimic the fine balance of phospholipid compositions and contents in real cell membranes. The highly regulated lipid composition of natural bacterial cell membranes is a vital component in the evaluation of ABP-membrane interactions. Thus, the diversity of bacterial membranes requires more appropriate stationary phases that can take account of their specific lipid compositions. Capillary electrophoresis (CE) has been used as an effective tool to investigate the specific or non-specific interactions between bioactive components and their targets [14–15]. Liposomes have been used as a pseudo-stationary phase in CE for the analysis of analyte-membrane interactions using peak shift methods. By supplementing the background electrolyte with a series of concentrations of liposomes, variations in the mobility of the bioactive component can be measured [16]. This method has the advantages of high analysis efficiency, low sample volume and low reagent consumption. The liposomes can disperse freely in the running buffer, which involves few steric restrictions and better mimics the interaction between bioactive components and lipid vesicles [17]. In a previous study, we developed an effective method of Escherichia coli cell membrane chromatography for the separation of ABPs, based on the affinity interaction between ABPs and bacterial cell membrane liposomes [18]. Detailed information regarding the interaction between endogenous bacterial membrane lipids and ABPs may be useful for screening and designing ABPs. The aim of this study was to investigate the interaction between E. coli membrane lipid liposomes and the antibacterial peptide Apep10 using the liposome as a pseudo-stationary phase in CE and ILC. The binding constant and thermodynamic parameters of Apep10 interacting with the E. coli membrane lipid liposome were also compared with those of the peptide with the specific mixed phospholipid liposome to analyse the interaction difference. Materials and Methods Chemicals Egg-yolk phosphatidylcholine (EYPC) and 1, 2-Dimyristoyl-sn-glycero-phosphatidylglycerol (DMPG) were purchased from A. V. T. Pharmaceutical (Shanghai, China). The antibacterial peptide Apep10 (GLARCLAGTL), screened from boiled-dried anchovies by immobilized bacterial membrane liposome chromatography, was provided by the School of Food Science and Engineering, Qingdao Agricultural University. Escherichia coli ATCC 25922 was obtained from the China Center of Industrial Culture Collection (CICC, Beijing, China). Luria-Bertani (LB) broth and agar medium were purchased from Baisi Biotechnology Co., Ltd. (Hangzhou, China). All other chemicals were of analytical grade and used without further purification. Extraction of E. coli membrane lipid E. coli was cultured aerobically in LB broth at 37°C, which is the optimal growth (...truncated)