br Conflict of interest br

Conflict of interest
Acknowledgments This work was supported by the US Army Research Office, grant number: W911NF-10-1-0150, US Army Research Office and MIT-ISN HBCU-MI program, US Army Centre of Environmental Health Research (USACEHR), contract number: W81XWH-11-C-0026, and the Romanian National Authority for Scientific Research, CNDI–UEFISCDI project number 25 PN-II-PT-PCCA-2011-3.2-1682. This work is also supported by the Malaysian Science Fund (SF12-019-0048) and Prototype Research Grant (PRGS 12-010-0010). The content of the information does not necessarily reflect the position or the policy of the Government or MIT, and no official endorsement should be inferred.
Introduction The plasma membranes of Gram-positive bacteria are surrounded by a layer of peptidoglycan, which is in the form of a rigid polymer network, providing structural strength and rigidity to support the bacteria against lysis [1]. Glycopeptide or are active against a wide range of Gram-positive bacteria thanks to their ability to bind peptidoglycan precursors (lipid II) with terminal sequence -Lys-d-Ala-d-Ala and prevent peptidoglycan synthesis, resulting in bacterial cell lysis [1,2]. Unfortunately, overuse and misuse of antibiotics has led to the emergence of resistance [3]. Vancomycin is a ‘last resort’ antibiotic against serious infections caused by Gram positive bacteria. Resistance to vancomycin is caused by a deceptively simple change of the terminal d-Ala of the peptidoglycan precursors with d-Lac, resulting in a 1000-fold decrease in ligand affinity [4]. In order to combat bacterial resistance, extensive efforts have been focused on gaining a deeper insight of the mode of action of antibiotics by means of studying the interactions between antimicrobial agents and model membranes [5–7]. Supported lipid membrane models have attracted great attention as they preserve many biophysical properties of cellular membranes [7]. Particularly, supported lipid bilayers (SLBs) and supported lipid monolayers (SLMs) have been widely utilized in combination with optical biosensors to analyze interactions with many varied membrane–ligand–analyte systems [8]. We have previously employed bacterial peptidoglycan precursors analogues N-α-Docosanoyl-ε-acetyl-Lys-d-Alanine-d-Alanine (doc-KAA) anchored in the SLMs together with SPR as model membranes to study the binding interactions between vancomycin group of glycopeptide antibiotics and mucopeptide analogue ligands anchored to lipid surface [9,10]. However, it has been reported that SLBs formed on an amphipathic polymer cushion (Biacore, L1 chip) demonstrated more advantages: (a) flexible and soft polymer cushions provide a lubricating layer between the surface and the membrane and maintain sufficient mobility for the lipid molecules [11]; (b) they can properly mimic the inherently complex nature of two-dimensionally fluid plasma membranes and do investigation of biological processes at the cellular level [12,13] and resist nonspecific adsorption [8]. Hence, in this study, we have employed doc-KAA incorporated SLBs as model membranes for binding analysis. An L1 chip (Fig. 1a) was used and two glycopeptide antibiotics vancomycin and teicoplanin (Fig. 1c) were tested. In addition, the cell membranes of Gram-positive bacteria contain more than one of the phospholipids types, such as phosphatidylglycerol (PG) with negative charge and zwitterionic phospholipids phosphatidylcholine (PC) [14]. Charge has previously been shown to influence antibiotic activity [15–17]. In this study, we have explored interactions between model membranes and antibiotics using SPR. Cantilever sensors are a unique label free technology in the sense that they can measure in-plane nanomechanical surface stress, which is not purely mass dependent [18–20]. When specific biochemical interactions occur between a ligand immobilized on one side of a cantilever and a target analyte in solution, a mechanical bending of the cantilever can occur due to a change in surface stress [18,19]. Recently, researchers have investigated the use of cantilever sensors to understand the biophysical forces involved in drug action [20–25]. Ndieyira and co-workers have reported the detection of vancomycin binding to bacterial cell wall precursor analogues on cantilever arrays and quantified binding constants for vancomycin-sensitive and vancomycin-resistant mucopeptide analogues [18]. However, this work was carried out with a rigid, polycrystalline self-assembled monolayer model of the cell wall mucopeptide. Liu et al. demonstrated that cantilevers can sense the formation of SLBs on a surface and observe mechanical properties changing of SLBs [19]. In order to gain insight into the mechanical properties of antibiotic–membrane interactions and a putative role in cell lysis, we have used cantilever sensors to observe stress change between glycopeptide antibiotics and more mimetic model membrane background (Fig. 1b).