Disruption of Asymmetric Lipid Bilayer Models Mimicking the Outer

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Disruption of asymmetric lipid bilayer models mimicking the outer membrane of Gram-negative bacteria by an active plasticin. Jean-Philippe Michel, Yingxiong Wang, Irena Kiesel, Yuri Gerelli, and Veronique Rosilio Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02864 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017

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Disruption of asymmetric lipid bilayer models mimicking the outer

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membrane of Gram-negative bacteria by an active plasticin

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J.P. Michel*a,b, Y.X. Wang a,b, I. Kiesel c, Y. Gerelli c and V. Rosilio a,b

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a

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Clément, F-92296 Châtenay-Malabry, France;

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b

CNRS, UMR 8612, F-92296 Châtenay-Malabry, France;

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c

Institut Laue-Langevin, 71 avenue des Martyrs, 38000, Grenoble, France.

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* Corresponding author: [email protected]

Univ Paris Sud, Institut Galien Paris Sud, Université Paris-Saclay, 5 rue Jean-Baptiste

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Abstract

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The outer membrane (OM) of Gram-negative bacteria is a complex and asymmetric bilayer

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that antimicrobial peptides must disrupt in order to provoke the cell lysis. The inner and

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external leaflets of the OM are mainly composed of phospholipids (PL), and

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lipopolysaccharide (LPS), respectively. Supported lipid bilayers are interesting model systems

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to mimic the lipid asymmetric scaffold of the OM and determine the quantitative and

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mechanistic effect of antimicrobial agents, using complementary physicochemical techniques.

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We report the formation of asymmetric PL/LPS bilayers using the Langmuir

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Blodgett/Langmuir Schaefer technique on two different surfaces (sapphire and mica) with

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synthetic phospholipids constituting the inner leaflet and bacteria-extracted mutant LPS the

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outer one. The combination of neutron reflectometry and atomic force microscopy techniques

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allowed the examination of the asymmetric scaffold structure along the normal to the interface

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and its surface morphology in buffer conditions. Our results allow discrimination of two

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structurally-related peptides, one neutral and inactive, and the other cationic and active. The

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active cationic plasticin PTCDA1-KF disrupts the asymmetric OM at relevant concentrations

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through a carpeting scenario characterized by a dramatic removal of lipid molecules from the 1 ACS Paragon Plus Environment

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surface.

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Keywords: Gram-negative bacteria; asymmetric bilayer; lipopolysaccharide; plasticin;

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surface pressure; neutron reflectometry, Atomic Force Microscopy; carpeting.

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Abbreviations: OM: outer membrane; PL: phospholipids; LPS: lipopolysaccharide; BAM:

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Brewster angle microscopy; DLS: dynamic light scattering; SOPE:1-stearoyl-2-oleoyl-sn-

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glycero-3-phosphoethanolamine; SOPG: 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-

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glycerol); CL: 1'.3'-bis[1.2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol; LE: liquid expanded

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state, LC: liquid condensed state; Kdo: 3-deoxy-D-manno-2-octulosonic acid, LB: Langmuir-

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Blodgett; LS: Langmuir-Schaefer; NR: neutron reflectometry; AFM: Atomic Force

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Microscopy; cac: critical aggregation concentration; SLD: scattering length density; MLV:

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Multi lamellar vesicles; MIC: Minimum inhibitory concentration.

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1. Introduction

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Various secretions of animal or invertebrate organisms contain antimicrobial peptides among

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which a majority possesses antibacterial activity. Antimicrobial peptides cause bacterial

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membrane permeabilization, leakage of the intracellular content, and osmotic instability

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resulting in bacteria death. In the case of Gram-negative bacteria, peptide–lipid interactions

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play a major role in the disruption process of the cell wall.1 Most active antimicrobial peptides

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are cationic.2 Also, many of them adopt α-helical structures favoring their interactions with

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phospholipids.3 However both features - charge and α-helical structure - are insufficient to

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fully explain peptide lytic activity.4

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It has been generally observed that above a minimum inhibitory concentration (MIC),

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accumulation of a peptide at a membrane surface initiates a process that affects membrane

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integrity, eventually leading to its lysis. Three models - carpet-like, pore formation (toroidal

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or worm-hole) and barrel-stave have been proposed to describe the mechanism of lipid

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membrane disruption.5 They have been grouped altogether under the name of Shai-

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Matsuzaki-Huang or SMH model.6 Yet, their experimental confirmation is still lacking so far,

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except for some peptides such as melittin, protegrin or alamethicin that induce barrel stave-

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shaped pores.7-9 Artificial lipid monolayers and bilayers are interesting tools for this purpose.

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In contact with Gram-negative bacteria, the peptides must first cross the outer membrane, a

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complex asymmetric bilayer, whose inner leaflet is essentially constituted of phospholipids

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(PL) such as phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL),

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whereas the external leaflet is made of lipopolysaccharide (Smooth LPS). S-LPS molecules

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are composed of a particular lipid (lipid A) attached to an O-specific polysaccharide chain.

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Lipid A is a phosphorylated diglucosamine molecule with five to seven covalently attached

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acyl C14-C18 chains, depending on the bacterial species.10 The O-specific polysaccharide chain

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possesses a high variability across bacterial strains and is an endotoxin that activates the

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immune system when released by bacteria lysis.11 Some bacteria are genetically modified to

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produce LPS with truncated oligosaccharide regions.12 The deep rough mutant LPS Re 595 is

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the shortest LPS molecule available, with both its outer core oligosaccharide and O-antigen

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regions truncated, leaving only the two sugars 3-deoxy-D-manno-octulsonic acid (Kdo) linked

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to the lipid A glucosamine headgroup.13

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Former works have described the formation, structure and physicochemical properties of

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LPS-rich bilayer models of the outer membrane, in the form of vesicles or stacked bilayers,

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using fluorescence microscopy and neutron scattering techniques.14-17 Wacklin and coworkers

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showed that the supported bilayers spontaneously formed by adsorption-fusion of two-

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phospholipid vesicles, were asymmetric by preferential adsorption of the lipid with the

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highest phase transition temperature to the solid/liquid interface.18-19 The asymmetric


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distribution of lipids in a bilayer may also result from electrostatic repulsion between charged

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phospholipids and the negatively charged hydroxyl groups of the silicon surface. For

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example, it has been observed that in POPC/POPS bilayers, the anionic POPS was detected in

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the outer bilayer leaflet only.20 However, in these planar bilayers, the lipid composition of

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each leaflet could not be tuned precisely.

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Combination of Langmuir Blodgett (LB) and Langmuir Schaefer (LS) transfers of floating

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monolayers allowed formation of asymmetric suspended 21-23or supported PL/PL and PL/LPS

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bilayers in a more controlled manner and their characterization by neutron reflectometry

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(NR).24-26 Atomic Force Microscopy (AFM) and fluorescence microscopy using labeled lipids

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allowed visualizing the organization of lipid molecules inside the asymmetric geometry, and

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the rearrangements occurring between the two leaflets of asymmetric PL/PL bilayers.27-28

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Several methods have been used to determine the mechanisms of antimicrobial peptide

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activity29, such as calorimetry measurements to study peptide effect on lipid

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thermodynamics30, fluorescence spectroscopy to observe the permeabilization of membrane

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vesicles7, electrical conductance measurements to assess the formation and stability of

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peptide-induced pores in suspended asymmetric bilayers31, circular dichroïsm or solid state

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NMR to measure the orientation and secondary structure of peptide bound to a lipid bilayer.32

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Wang et al. used the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to

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characterize the disruption process of egg-PC symmetric bilayers by the antimicrobial peptide

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chrysophin-3. Analysis of the QCM-D signals showed that this peptide acted against the

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membrane through a pore formation process.33 Peptide-induced domain formation in

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supported symmetric lipid bilayers was demonstrated by combination of AFM and polarized

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total internal reflection fluorescence microscopy.34 More recently, a study using NR

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highlighted the role of the saccharide moieties of the LPS outer core region in the screening of

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electrostatic interactions between the LPS phosphate-rich inner core region and the


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antibacterial protein colicin adsorbed onto the LPS leaflet.35 It would be of great interest to

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examine the asymmetric PL/LPS bilayer structure after interaction with a molecule able to

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penetrate separately the two leaflets of the OM.38

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We have studied two model peptides, one active and the other inactive, belonging to the

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dermaseptin family. PTCDA1 is a natural neutral plasticin, extracted from skin exudates of

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Pachymedusa dacnicolor frog. Its sequence consists of 23 amino acids. It has no antimicrobial

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activity on conventional laboratory strains.36 The peptide PTCDA1-KF results from the

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modification of PTCDA1, by replacement of amino acids at positions 8 and 12 with lysines

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(K) and substitution of the amino acid at position 18 with a phenylalanine (F). These changes

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in the sequence confer a net positive electrical charge (+2) to this peptide. PTCDA1-KF

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shows very good antimicrobial activity on Gram-positive and Gram-negative strains.37

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However, its mechanism of action is still unknown. In a previous work38, we showed that both

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plasticins adsorb at the air/solution interface, and exhibit the same critical aggregation

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concentration (cac = 0.14 ± 0.01 µM). The cationic peptide however, lowers slightly more the

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surface tension than the neutral one. We characterized the first steps of the interaction of these

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plasticins with bacteria OM using a LPS Re 595 mutant/S-LPS mixture mimicking the

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external leaflet, and a SOPE/SOPG/cardiolipin one mimicking the inner lipid layer.38 We

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showed that upon interaction with LPS liposomes, the cationic plasticin achieved partial

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disaggregation and formed oligomers of α-helices, whereas the native one remained

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aggregated and unstructured. Peptide insertion experiments and grazing incidence X-ray

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diffraction demonstrated the effective penetration of PTCDA1-KF into condensed mixed LPS

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monolayers, and the consequent complete loss of organization of these monolayers.38

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In the present article, the interaction of the two plasticins with asymmetric PL/LPS bilayers

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formed by LB/LS transfer was investigated by NR and AFM. The mutant LPS Re 595

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extracted from S. enterica was chosen for its good ability to form monolayers and liposomes.


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We focused on the influence of peptide nature and concentration on a plasticin penetration

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mechanism and alteration of the asymmetric bilayer structure.

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2. Material and Methods

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2.1 Material

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PTCDA1 (GVVTDLLNTAGGLLGNLVGSLSGNH2, Mw = 2125 g/mol) and PTCDA1-KF

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[K8,12, F18]-Plasticine-DA1 (GVVTDLLKTAGKLLGNLFGSLSGNH2, Mw = 2260 g/mol)

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were synthetized at the Plateforme d’Ingénierie des Protéines et Synthèse Peptidique (Ch.

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Piesse, IFR 83, UPMC, France).

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The

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(18:0-18:1 PE or SOPE, Mw = 746.05 g/mol), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-

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rac-glycerol) (sodium salt) (18:0-18:1 PG or SOPG, Mw = 799.05 g/mol), and 1'.3'-bis [1.2-

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dioleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt) (18:1 CL, Mw = 1501.98 g/mol), as

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well as the partially deuterated 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphoethanolamine

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(16:0-18:1 PE-d31 or POPE-d31, Mw = 749.19 g/mol) and 1-palmitoyl-d31-2-oleoyl-sn-

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glycero-3-[phospho-rac-(1-glycerol)] (16:0-18:1 PG-d31 or POPG-d31, Mw = 802.18 g/mol)

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were purchased from Avanti Polar Lipids (Alabaster, AL). They were 99% pure and were

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used without further purification. The deep rough mutant strain lipopolysaccharide (LPS Re

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595 mutant from source strain 49284, Mw = 2500 g/mol) was obtained from Sigma-Aldrich,

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produced from phenol-chloroform-petroleum ether extraction from Gram-negative bacteria

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Salmonella enterica serotype minnesota. LPS Re 595 is denoted as ‘LPS’.

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Sodium chloride (NaCl, 99% pure, Mw = 58.44 g/mol), sodium phosphate monobasic

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monohydrate (NaH2PO4.H2O, 98% pure, Mw = 137.99 g/mol), sodium phosphate dibasic

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heptahydrate (Na2HPO4.7H2O, 98% pure, Mw = 268.07 g/mol) and sodium dodecyl sulfate

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(SDS, Mw = 288.38 g/mol) were purchased from Sigma-Aldrich. Chloroform and methanol

hydrogenated

phospholipids

1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine


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(99% pure) provided by Merck (Germany) were analytical grade reagents, and were used

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without further purification. The buffer was a phosphate-buffered saline (PBS) solution with

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100 mM sodium phosphate and 150 mM NaCl at pH 7. Peptide powders were dissolved in

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buffer with concentrations ranging from 0.05 mM to 1 mM. Ultrapure water (γ = 72.2 mN/m

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at 22°C, resistivity 18.2 MΩ.cm) was produced by a Millipore MilliQ Direct 8 apparatus.

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Prior to the experiments, all glassware was soaked for an hour in a freshly prepared hot TFD4

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(Franklab, Guyancourt, France) detergent solution (15% v/v), and then thoroughly rinsed with

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ultrapure water.

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2.2 Methods

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Formation of lipid monolayers

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Surface pressure-area (π-A) measurements of monolayers of the pure phospholipids (SOPE,

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SOPG and CL), their mixture in a 80/15/5 molar ratio, and the pure mutant LPS were

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performed using a thermostated Langmuir film trough (775.75 cm2, Biolin Scientific, Finland)

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enclosed into a Plexiglas box to limit surface contamination. Solutions of phospholipids and

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LPS in chloroform-methanol 9:1 v/v were spread onto buffer. Prior to monolayer spreading,

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the subphase surface was cleaned by suction. After spreading, a waiting time of 20 minutes

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allowed complete evaporation of the organic solvents. Compression was then performed at a

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speed of 5 Å2×molecule-1×min-1. All experiments were performed at 21°C, and the results

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reported are mean values of at least three measurements.

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Analysis of π-A isotherms

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To assess the relative average area variations induced by addition of SOPG and CL to SOPE,

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the experimentally determined molecular areas of the mixed monolayers (AEXP) were

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compared to the corresponding theoretical ones (ATH) calculated according to the additivity 7 ACS Paragon Plus Environment

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rule: ATH = XPE APE + XPG APG + XCL ACL, where X stands for the molar fraction of the

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various phospholipids. The excess molecular areas ∆AEXC were calculated from the difference

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AEXP-ATH. Negative deviations from the additivity rule correspond to monolayer condensation

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and may indicate intermolecular accommodation and/or dehydration interactions between

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lipids in a mixed film.39 Positive deviations from the additivity rule are the result of area

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expansion and would account for unfavorable interactions between the different components

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leading to poor mixing.

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From the surface pressure-area data, the surface compressional moduli K of monolayers were

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calculated, using Eq. 1:

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 dπ  K = −A   (Eq. 1)  dA T

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According to Davies and Rideal40, K values below 12.5 mN/m, in the range 13-100 mN/m,

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100 to 250 mN/m and above 250 mN/m would account for the gaseous state, the liquid

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expanded state (LE), the liquid condensed state (LC) and the solid state of a monolayer,

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respectively.

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LB/LS transfers of lipid monolayers on mica or sapphire

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Langmuir-Blodgett (LB) film transfer was performed on the Langmuir trough equipped with a

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polytetrafluoroethylene well. Mica or sapphire surfaces were attached to the dipper for LB

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transfer after cleaning process (cleavage for mica or piranha treatment for sapphire). They

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were immersed into buffer subphase, and then the ternary phospholipid mixture solution was

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spread onto the subphase. After solvent evaporation, the monolayer was compressed until the

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desired surface pressure (40.0 ±1.0 mN/m) was reached. When the surface pressure remained

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constant (± 0.2 mN/m), the substrate was slowly lifted from the subphase at the speed of 1.0


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mm/min, while the surface pressure was maintained at 40.0 mN/m. The monolayer-bearing

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surface was examined by AFM or used for further monolayer transfer. The transfer of the LPS

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monolayer at the surface pressure of 40 mN/m, using the LS (Langmuir-Schaefer) method,

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was performed immediately after LB transfer of the phospholipid monolayer on the mica or

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sapphire surface. The surface was dipped through the interface and lowered into the well of

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the trough to keep the sample in aqueous conditions before the transfer into the measurement

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AFM or NR cell. All transfers were done at 21°C.

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Liposomes preparation and DLS measurements

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Liposome formulations made of either pure LPS or SOPE/SOPG/CL (80/15/5) were prepared

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according to the Bangham’s method followed by vesicle extrusion.14, 41 LPS was dispersed

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into a mixture of chloroform and methanol (9:1 v/v) at a concentration of 1.0 mg/ml (0.4

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mM). The PL mixture was prepared in the same solvent mixture at 16 mM. Both organic

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solutions were evaporated for 3 hours at 60°C under reduced pressure, and the resulting dry

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lipid films were hydrated with buffer and sonicated for a few minutes. The obtained LPS or

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SOPE/SOPG/CL suspensions were used for DSC measurements.

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Differential scanning calorimetry (DSC) experiments

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In order to determine the gel/fluid transition temperature of pure LPS and the PL mixture,

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DSC measurements were performed with a VP-DSC calorimeter (Microcal) at the IBBMC lab

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(Univ Paris Sud, UMR8619, Orsay, France) using 200 nm MLV at 2.5 mg/ml with the

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aforementioned lipid composition. Samples equilibrated at 20°C for 15 min were then heated

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up to 60°C at a constant rate of 1°C/min. Three successive scans were acquired for each


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sample. Data were analyzed using the MicroCal Origin software provided by the

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manufacturer.

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Atomic Force Microscopy experiments

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AFM experiments were performed using the Nanowizard 3 Ultra Speed from JPK Instruments

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(Berlin, Germany, www.jpk.com), installed on an air-buffered table coupled to a dynamic

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anti-vibration device, and enclosed in an acoustic box. Because of its plate like structure

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composed of an octahedral alumina sheet sandwiched by two tetrahedral silicate sheets, the

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cleavage of muscovite mica surface reveals a molecular smooth surface over micrometric

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areas. Mica substrates with surface area of 1.5x1.5 cm2 were used (Ted Pella Inc, Redding

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USA). Sapphire disks of 12 mm diameter were provided by Bruker (Bruker Nano Surfaces,

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Palaiseau, France). Their surface roughness was checked by AFM in air after piranha

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treatment and was less than 500 pm. We used the same procedure for the monolayer transfers

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as described in the previous section. The only difference lies in the fact that the buffer

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subphase used for the LB transfer of the anionic SOPE/SOPG/CL monolayer on the mica

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surface was complemented with 1mM CaCl2 to create a layer of divalent cations adsorbed

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onto the solid surface, which bridged the anionic PL headgroups during monolayer transfer, as

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previously demonstrated for PC/PG bilayers.42-44

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Imaging of asymmetric bilayers or LB-transferred PL monolayers on their substrates (mica or

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sapphire) was performed in buffer (in air for monolayers) in AC-HyperDrive® mode, with

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gold coated silicon cantilevers PPP-NCHAuD of 40 ± 10 N/m spring constant and 290 ± 5

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kHz resonance frequency (Nanosensors, Neuchatel, Switzerland). The pyramid-shaped tips

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had a radius of curvature less than 10 nm. A free amplitude oscillation of 1 nm was chosen

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allowing a very high resolution of the imaged surface. Images were taken at scan rates of 1 or

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2 Hz. Image processing (flatten, plane fit, edge and hole detection) was performed with the 10 ACS Paragon Plus Environment

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JPK Data Processing software (JPK Instruments). At least three different areas of each sample

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were scanned and typical images were presented. Average values of height and lateral

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dimensions of domains or holes were determined with all domains or holes detected in the

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pictures. The morphology of the bilayer was examined after incubation with a peptide for 30

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min and then rinsing. Force measurements were performed with the same cantilevers at the

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speed of 1 µm/s in a Z-closed loop over 1 µm with setpoints around 500 nN.

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Neutron reflectometry

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Neutron reflectivity experiments were performed at the sapphire/buffer interface on the

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FIGARO reflectometer at the Institut Laue-Langevin (ILL, Grenoble, France).45 Time-of-

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flight measurements were performed using wavelengths in the range of 2 to 30 Å with two

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different angular configurations allowing Q values to range from 0.005 to 0.2 Å-1. For a given

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incident wavelength λ and angle αi, the component of the wave-vector transfer Q in the

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direction perpendicular to the layer interface is Q = 4π/λ sin αi.

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Sapphire single crystals cut along the (111) plane were first cleaned by immersion in a freshly

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prepared piranha solution for 30 min, then dried under a clean nitrogen stream before a

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UV/ozone treatment for 45 min. They were stored in ultrapure water until bilayer deposition.

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As the sapphire surface is naturally positively charged in aqueous buffer, no functionalization

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was required for LB transfer of the negatively charged POPE-d31/POPG-d31/CL monolayer

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(80/15/5). Sample holders were laminar flow cells that allowed the solvent exchange to apply

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the contrast variation method.46 High-purity D2O (ILL) and Milli-Q water were used in

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different ratios to obtain media with scattering-length densities (SLDs values) of 6.35, 3.00,

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1.00 and -0.56 (× 10-6 Å-2).

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Solid-supported asymmetric PL/LPS bilayers were deposited using the NIMA trough

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available at the Partnership for Soft Condensed Matter (ILL, Grenoble) using the LB/LS 11 ACS Paragon Plus Environment

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methods and according to the same procedure as described above for the LB transfer of the

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mixed POPE-d31/POPG-d31/CL monolayer at the surface pressure of 40 mN/m on sapphire

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surface. As fully deuterated molecules, such as 16:0 PE-d62 or 18:0 PE-d70 (the same for PG),

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do commercially exist but only in saturated forms, partially deuterated POPE (16:0-18:1 PE-

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d31) and POPG (16:0-18:1 PE-d31) molecules were chosen to form the phospholipid mixture,

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resulting in a higher scattering contrast between them and the hydrogenated LPS. The transfer

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of the LPS monolayer using the LS method was performed immediately after the LB transfer

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of the phospholipid monolayer onto the sapphire surface. The surface was dipped through the

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interface and lowered into the well of the trough onto the flow module of the solid-liquid cell

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used for the NR experiments. Cells were filled with buffer and sealed directly in the well.

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Reflectivity R can be defined as a function of the variation of the neutron scattering length

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density (SLD) along the direction perpendicular to the sample surface.47 In the case of a

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multi-interfaces system, the total reflected intensity arises from the sum of the neutrons

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reflected by each interface. Parratt’s recursion relation48 is an iterative exact method allowing

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the calculation of the reflectivity for a multi-layered system. The sample is divided into

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several layers and the components of the reflected and transmitted beams are calculated

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recursively. Because of this dynamical aspect of the method, multiple scattering effects as

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well as self-adsorption are taken into account. Each layer is described by four parameters

291

(thickness, SLD, water content and roughness). Data were analyzed using the Aurore

292

software49 that allows the simultaneous analysis of curves measured in the different contrast

293

configurations. The number of free parameters present in the model can be reduced by

294

imposing constraints on the scattering length densities and thicknesses as described in ref 49

295

and references therein. For asymmetric PL/LPS bilayers formed on sapphire surface, the SLD

296

profile was modeled by five layers from sapphire surface to the bulk solution: water layer,

297

inner phospholipid headgroup layer, inner phospholipid acyl chains layer, outer LPS acyl


Page 13 of 39

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298

chains layer, and outer LPS Re 595 headgroup layer. Confidence intervals in the calculated

299

reflectivity profiles and in the corresponding SLD profiles were obtained by the use of a

300

bootstrap method described in ref 49. This method computes the statistical distribution for

301

every free parameter, taking into account correlations effects. Errors on single parameter

302

values are calculated from the width of these distributions. Plasticin injection was followed by

303

an incubation time of 30 min before rinsing the excess material.

304 305

3. Results

306

3.1 Lipid monolayers mimicking each leaflet of a bacteria outer membrane

307

Compression isotherms of the SOPE/SOPG/CL mixture at the 80/15/5 molar ratio and the

308

pure mutant LPS Re 595 spread at the air-buffer interface are presented in Figure 1. The

309

isotherms of pure lipids and characteristic values are summarized in the Figure S1 of the

310

Supporting Information. Both phospholipid mixture and pure LPS formed stable monolayers

311

at the air/buffer interface, up to surface pressures of 50 mN/m.51-52

312

The negative excess molecular areas (∆AEXC) for the SOPE/SOPG/CL mixture accounted for

313

attractive interactions between the film-forming components. This monolayer was in a liquid-

314

expanded state over the whole surface pressure range (compressibility moduli K < 100 mN/m,

315

see inset to Figure 1), and appeared completely homogeneous upon compression as verified

316

by Brewster angle microscopy measurements. No nuclei of condensation nor stripe was

317

observed except at collapse (π > 50 mN/m, data not shown).

318

Comparison of the isotherms shows that at 40 mN/m, LPS and phospholipid molecules

319

occupy 154 Å2 and 57 Å2, respectively. The LPS monolayer is much more expanded than the

320

PL one due to the larger size of its complex polar head but especially its five-to-seven

321

hydrocarbon chains. However, it is also the most rigid monolayer as deduced from the

322

compressibility modulus-surface pressure relationships (inset to Figure 1). 13 ACS Paragon Plus Environment

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Page 14 of 39

323

324 325

Figure 1: surface pressure-area isotherms of the SOPE/SOPG/CL mixture and pure LPS

326

spread at the air/buffer interface. Inset: Compressibility modulus versus surface pressure for

327

the two monolayers.

328 329

3.2 Characterization of the PL monolayer by Atomic Force Microscopy

330

In order to get a better insight into the organization of the lipid mixture adsorbed at the

331

surface of the substrates used in NR and AFM experiments, we transferred the PL mixture

332

onto mica (Fig. 2A) and sapphire surfaces (Fig. 2B) by LB transfer at 40 mN/m, and observed

333

the morphology of the monolayers by AFM in air.

334

Figure 2A shows a discontinuous monolayer, as inferred from the presence of 2.0 (± 0.2) nm-

335

deep holes consistent with the thickness of a PL monolayer with C18 chains, and a lateral

336

dimension of 231 ± 40 nm. These holes represent 8 to 10% of the surface, indicating that 90%

337

of the mica surface was covered by the phospholipid monolayer. The surface roughness of the

338

PL chains is 210 pm. 14 ACS Paragon Plus Environment

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339

340 341

Figure 2: AFM height image in air of the SOPE/SOPG/CL monolayer after transfer onto a

342

mica surface (A) and sapphire surface (B) at the surface pressure of 40 mN/m.

343 344

Figure 2B also shows holes, but with a much smaller lateral dimension (22 ± 8 nm). These

345

holes represent 2 to 3% of the surface only, accounting for the coverage of 97% of the

346

sapphire surface by the monolayer. The surface roughness of the pointing up PL chains is

347

slightly higher (250 pm) than that observed on mica.

348 349

3.3 Supported asymmetric bilayers

350

3.3.1 Imaging of asymmetric PL/LPS supported bilayers on mica surface by Atomic Force

351

Microscopy

352

Figures 3A and 3B show the surface morphology (height and phase) in buffer of the

353

asymmetric PL/LPS bilayer formed onto mica by LB/LS deposition. They reveal a relatively

354

flat surface at the nanoscale level. The bilayer is uniform and covers the whole surface, but as

355

previously observed for the monolayers, it is not continuous. Small dark circular domains

356

coexist with a surrounding lighter matrix (Figure 3C). The spatial extension of these dark

357

circular domains is 286 ± 18 nm in average, and represents 3-4% of the bilayer surface area. 15 ACS Paragon Plus Environment

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Page 16 of 39

358

Their depth is 5.1 ± 0.3 nm in average (Figure 3E). These domains correspond to holes in the

359

matrix.

360

361 362

Figure 3: AFM height (A) and phase (B) images of the asymmetric PL/LPS bilayer. Height

363

image of holes (C) and height profiles (D, E) performed along the black arrows in C. Z-scales

364

are shown on the right side of each picture.

365 366

The corresponding phase image of the asymmetric bilayer shows a lower phase shift in the

367

holes than in the surrounding matrix (Figure 3B), indicating a more rigid phase in the holes

368

than in the higher phase: the bottom of the holes is very likely the mica surface. The PL/LPS

369

bilayer is thus slightly porous and the matrix has a roughness varying between 300 and 400

370

pm (Figure 3D). The force-volume picture corresponding to Figure 3A (Fig. S3 in the


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371

Supporting Information) confirms the uniformity of the PL/LPS bilayer on mica and the

372

presence of holes.

373 374

3.3.2 Analysis of asymmetric PL/LPS bilayers on sapphire surface by neutron reflectometry

375

We measured the reflectivity on cleaned blocks in 4 different contrasts (H2O, D2O, 1MW and

376

3MW with SLD -0.56, 6.35, 1.00 and 3.00 (× 10-6 Å-2) respectively). For a given contrast, the

377

reflectivity curves for the two sapphire blocks were identical, confirming the same surface

378

state for the two blocks. Surface roughness of sapphire surfaces was then fixed at 3Å for

379

further measurements.

380

Figures 4A and 4B show the neutron reflectivity experimental curves, model fits and the

381

resulting SLD profiles for the asymmetric PL/LPS bilayer. The experimental curves were

382

successfully fitted to a five-layer model describing, from sapphire surface to the bulk solution,

383

a water layer (1st layer), the inner phospholipid leaflet headgroups (2nd layer), the inner

384

phospholipid leaflet acyl chains (3rd layer), the outer LPS leaflet acyl chains (4th layer) and the

385

outer LPS leaflet headgroups (5th layer). The measurement in 4 different contrasts allowed

386

revealing the fine composition of the bilayer since the contributions arising from the different

387

chain regions (PL and LPS chains) and polar head groups could be clearly distinguished. The

388

use of partially deuterated phospholipids (POPE-d31 and POPG-d31) allowed enhancement of

389

the contrast between the PL chains and the LPS ones. The SLD profiles (Figure 4B)

390

demonstrate the asymmetric nature of the interfacial structure with two leaflets of different

391

lipid composition, particularly in the hydrophobic region. For comparison, symmetric PL/PL

392

bilayers were also formed by LB/LS transfers and displayed symmetric SLD profile (Fig. S2

393

in Supporting Information).


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394 395

Figure 4: Neutron reflectivity experimental curves in RQ4 vs Q plot (symbols) measured in 4

396

different contrasts (A): H2O (○), D2O (□), 1MW (∇), 3MW (+), and fit profiles for an

397

asymmetric PL/LPS bilayer on sapphire surface. (B) Resulting SLD profiles and (C) model

398

parameters extracted from the fitting of experimental curves presented in (A).

399 400

The structural parameters obtained for the best fit of the experimental curves are shown in

401

Figure 4C. A 1Å-thick water layer was barely detected between the sapphire surface and the

402

first leaflet of the asymmetric bilayer. The hydrogenated PL headgroups of the inner leaflet

403

were found to be 7 ± 1 Å thick, a fully consistent value for PE or PG headgroups. The

404

associated SLD value was 1.36 ± 0.2x10-6 Å-2, identical to the one expected for a

405

POPE/POPG/CL mixture (calculation gives 1.36 x10-6 Å-2)53 and water content amounted to

406

69 ± 6%. This value is consistent with the thin underlying water layer and in agreement with

407

the typical values for hydrated phospholipid headgroups.25 The thickness of the mixed acyl


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408

chain layer was 11 ± 0.1 Å, slightly lower than the one expected for C16-C18 stretched acyl

409

chains. The corresponding SLD value (1.05 ± 0.07) x10-6 Å-2 appeared lower than expected

410

(calculation gives 2.23 x10-6 Å-2)53, suggesting that the PL/LPS bilayer was not fully

411

asymmetric.

412

The five-to-seven hydrogenated saturated acyl chains of LPS formed a 14 ± 0.5 Å-thick layer,

413

consistent with the length of stretched C14 hydrocarbon chains with a SLD value of (-0.50 ±

414

0.05) x10-6 Å-2, very close to the expected value (-0.29 x10-6 Å-2)53-54. Both layers of acyl

415

chains contained very low amount of water (3 ± 2% and 6 ± 1% for the PL and LPS chains

416

respectively), demonstrating the good integrity of the asymmetric bilayer on sapphire surface.

417

Finally, the hydrogenated LPS headgroups formed a 16 ± 2 Å-thick layer with a SLD value of

418

(2.2 ± 0.2) x10-6 Å-2 with 22 ± 1% water content. The difference in polar headgroups

419

thickness between the two leaflets confirms the asymmetry of the bilayer. The roughness (5 ±

420

2 Å) of the LPS headgroups layer was found slightly higher than the one determined by AFM

421

for a similar asymmetric bilayer deposited onto mica (Fig. 3D).

422 423

3.4. Interaction of plasticins with asymmetric PL/LPS bilayers

424

3.4.1. Interaction of PTCDA1 with the asymmetric PL/LPS bilayer by AFM

425

The state of the asymmetric bilayer following incubation with 1 µM PTCDA1 was examined

426

by AFM (Figures 5A and 5B). The bilayer integrity was preserved: no damage was detected

427

in the surface morphology of the upper leaflet.

428


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429 430

Figure 5: AFM height (A) and phase (B) images of the asymmetric PL/LPS bilayer on mica in

431

buffer after incubation with the neutral plasticin PTCDA1 at 1 µM. Height (C) and phase (D)

432

AFM pictures of another area of the bilayer after abundant buffer rinsing.

433 434

The dark areas in Figures 5A and 5B were initial defects in the bilayer. A few patches could

435

be detected on top of the bilayer. They appeared with a lower shift phase than the lipid matrix

436

indicating a more rigid material, probably adsorbed peptide aggregates. Their height was only

437

a few hundreds of pm and their shape was not well resolved despite many trials. They were

438

easily displaced by the AFM tip, indicating that they were weakly bound to the surface. Note

439

that during this experiment, the AFM laser signal detected on the photodiode was constantly

440

disturbed by the presence of peptide material in the aqueous volume, altering the resolution

441

and confirming the poor adsorption of PTCDA1 onto the asymmetric bilayer. Figures 5C and


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442

5D show another area of the PL/LPS bilayer after a thorough buffer rinsing, demonstrating the

443

preserved integrity of the bilayer and the absence of peptide patches in this area after rinsing.

444 445

3.4.2 Interaction of PTCDA1-KF with the asymmetric PL/LPS bilayer by AFM

446

Interaction between the cationic plasticin and the asymmetric bilayer was first monitored with

447

AFM at concentrations below its cac. Figures 6A and 6B show the resulting height and phase

448

images of the bilayer in buffer after 30 min interaction with PTCDA1-KF at 0.1 µM.

449

450 451

Figure 6: AFM height (A and phase (B) images of the asymmetric PL/LPS bilayer on mica in

452

buffer after incubation with PTCDA1-KF at 0.1 µM. (C) Height profile performed along the

453

black arrow in A. (D) Height image of individual patches. Z-scales are shown on the right

454

side of each picture.

455


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Page 22 of 39

456

Numerous patches were detected on the mica surface, demonstrating the loss of integrity of

457

the asymmetric bilayer (Figure 6C). Patches height was 2.0 ± 0.55 nm in average,

458

corresponding to that of a lipid leaflet, and their lateral dimension was 520 ± 35 nm (Figure

459

6E). They covered only 45 to 60 % of the mica surface area. These patches were associated

460

with a higher phase shift than the surrounding surface indicating that they were more

461

viscoelastic than the mica surface (Figure 6D). The tip did not displace them, despite

462

successive scans, indicating a strong adsorption to the surface. They are probably composed

463

of a mixture of lipids and peptide, although such information cannot be inferred from the

464

AFM images.

465

466 467

Figure 7: AFM height (A) and phase (B) images of the asymmetric PL/LPS bilayer after

468

interaction with PTCDA1-KF at 1 µM. Height and phase (C) images of the area contained in

469

the black square of image A. Z-scales are shown on the right side of each picture. (D) Height

470

profile obtained along the line (black arrow) in image C. (E) Force measurement obtained on

471

darker areas in image B (o for extend curve et ∆ for retract one). The slope of curves below

472

the x=0 position (contact point between AFM tip and surface) was calculated at 31.7 N/m.


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473

The effect of peptide incubation with the bilayer is even more striking when the concentration

474

of the peptide is raised to 1 µM (Figures 7A and 7B). The asymmetric bilayer appears almost

475

wiped off the mica surface: only a few small white patches are detected, demonstrating that

476

the integrity of the bilayer is totally lost. Patches height is 2.0 ± 0.45 nm in average, their

477

lateral dimension 320 ± 55 nm (Figure 7D) and they cover only 10 to 15 % of the surface

478

area. They are surrounded by fragments and debris 200 to 400 pm in height, which are spread

479

all over the surface (Figure 7C). Force-distance measurements were performed between the

480

patches, on darker areas of Figure 7C, and they display full linear behavior with slope

481

corresponding to the pure deflection of the cantilever on mica (~31 N/m, Figure 7E),

482

demonstrating the absence of leaflet beneath the patches.

483 484

3.4.3 Interaction of PTCDA1-KF with the asymmetric PL/LPS bilayer by NR

485

NR allowed investigation of the structure of asymmetric PL/LPS bilayer following incubation

486

with the cationic plasticin at 25 µM, a concentration well above its cac and MIC. At such high

487

concentration dramatic structural perturbation of the bilayer was expected, which was indeed

488

observed. Figures 8A and 8B show the experimental curves measured in the 4 different

489

contrasts, model data fits and SLD profiles. The data could not be fitted with a six-layer

490

model. The fit was then done with a five-layer one. This indicates that, after a strong

491

adsorption of the cationic plasticin to the LPS leaflet,38 a 30 min incubation followed by

492

rinsing did not result in a persistent adsorption of the cationic peptide onto the bilayer. The

493

resulting SLD profiles appear severely altered in comparison with those obtained before

494

interaction (see Fig. 4B): the asymmetry was lost. The very high water contents in the LPS

495

headgroup and acyl chain layers (80-85%) confirmed the above results (Fig 8C). Drastic

496

changes were also noticed in the lower leaflet directly adsorbed onto the sapphire surface,

497

such as the dehydration of the polar PL headgroups. The thickness and the SLD value for PL


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Page 24 of 39

498

headgroups decreased, whereas that of mixed deuterated/hydrogenated chains increased.

499

However, the water layer between the PL leaflet and the sapphire was not correctly resolved

500

by the fit as inferred from the roughness similar to thickness. The lower leaflet was still

501

present but was severely altered as the upper one.

502

503 504

Figure 8: Neutron reflectivity experimental curves in R vs Q plot (symbols) and fit profiles

505

measured in 4 different contrasts (A): H2O (○), D2O (□), 1MW (∇) and 3MW (+) after

506

interaction of 25µM PTCDA1-KF with a PL/LPS bilayer adsorbed onto a sapphire surface.

507

The resulting SLD profiles are in (B) and the model parameters in (C).

508 509

4. Discussion

510

To mimic the outer membrane of Gram-negative bacteria, we built planar supported PL/LPS

511

asymmetric bilayers containing LPS in the upper leaflet and phospholipids in the lower one.

512

The formation and structural characterization of this asymmetric lipid matrix was monitored 24 ACS Paragon Plus Environment

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Langmuir

513

by NR and AFM in order to explore its structure, composition and surface morphology. Mica

514

was used as a surface in AFM experiments for its ability to reveal flat areas over the

515

micrometer range with a complex surface composition and a zwitterionic surface charge.

516

Sapphire was adapted to NR measurements due to its transparency to neutrons and its positive

517

surface charge at pH 7 necessary for effective LB transfer of the anionic phospholipid

518

monolayer.

519

AFM allowed exploring the surface morphology of the upper interface of the PL/LPS bilayer

520

on mica surface, determining the bilayer thickness (5.1 ± 0.3 nm) and roughness and revealing

521

discontinuities in its structure (leading to 97% surface coverage, a value in agreement with

522

previous works on silicon dioxide surface 26). The presence of holes in the bilayer has to be

523

related to the discontinuous structure of the inner PL leaflet on mica surface.

524

NR results confirmed the asymmetric structure of the PL/LPS bilayer and its uniformity on

525

sapphire surface as inferred from the very low amounts of water found in the PL and LPS acyl

526

chains region. The total thickness of the asymmetric PL/LPS bilayer on sapphire was 4.8 ±

527

0.2 nm, if we neglect the 1 Å-thick water layer, a value very close to that measured by AFM

528

of the same bilayer on mica. Previous studies using NR reported similar values for the

529

thickness, water content of acyl chains and SLD values of a PL leaflet 25 and Lipid A and kdo

530

units in a mutant LPS (Rc-LPS).16,26,54 Lakey’s group studied the structure of asymmetric

531

DPPC/Lipid A and DPPC/mutant LPS bilayers formed by LB/LS deposition at 27 mN/m on a

532

silicon surface.26 For the LPS polar headgroups, they reported thicknesses between 8 ± 5 Å

533

for Lipid A, and 20.9 ± 2 Å for Rc-LPS. The value of 16 ± 2 Å that we obtained for the LPS

534

Re 595 headgroup is therefore consistent with the differences in length between Lipid A, and

535

the two LPS mutants.

536

The degree of asymmetry of the bilayer is related to the relative proportion of dry matter of

537

lipid (PL or LPS) in each leaflet, corrected by the solvent fraction of the considered region.


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Page 26 of 39

538

Due to the minimal isotopic contrast between the phospholipid and LPS headgroups, only

539

volume fractions of phospholipid, LPS and water from the lipid tail regions of each leaflet

540

were considered. The proportion of PL in the inner leaflet was estimated to 62 ± 4 %,

541

accounting for partial asymmetry of the bilayer (with 9% water in the hydrophobic region).

542

This value is in agreement with previous works with asymmetry of phospholipid/LPS bilayers

543

around 65% PL in the inner leaflet (and 10% water), 26 but lower to that of asymmetric PL/PL

544

bilayers (> 90% dry matter and less than 10% water).25

545

The discrepancy between the expected and measured SLD values of the hydrophobic region

546

in the asymmetric PL/LPS bilayer (Fig. 4C) could be explained by two different facts. The

547

first reason could be the weak sensitivity of neutron reflectivity to the partial deuteration of

548

POPE and POPG in the inner leaflet. We have chosen 16:0-18:1 PE-d31 and 16:0-18:1 PG-d31

549

because both possess one deuterated saturated acyl chain and a hydrogenated one with one

550

insaturation to mimic the level of insaturation and chain length commonly found in Gram-

551

negative bacterial membranes.55 However, this labeling may not be sufficient to generate

552

enough contrast in NR measurements. Fully deuterated molecules, such as 16:0 PE-d62 or 18:0

553

PE-d70 (the same for PG), do commercially exist but only in saturated forms. The other cause

554

could be a minor lipid exchange between the two leaflets during NR experiments. However,

555

the bilayer structure was examined once again a few hours after initial NR measurement and

556

no noticeable change in the bilayer structure was observed. Significant exchange between the

557

inner and outer bilayer leaflets due to flip-flop mechanism25,49 is avoided because of the much

558

larger area occupied by one LPS molecule compared to that occupied by a PL one. Moreover,

559

using differential scanning calorimetry, we measured the phase transition temperatures of the

560

SOPE/SOPG/CL mixture and LPS Re 595. We found 25.16 ± 0.02°C and 25.97 ± 0.02°C,

561

respectively, indicating that at room temperature, both lipid systems were in the gel phase.

562

This gel phase behavior is also valid for the POPE/POPG/CL mixture used for NR 26 ACS Paragon Plus Environment

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563

measurements. Furthermore, monolayer transfers were all performed at 40 mN/m. At this

564

pressure, the pure LPS Re 595 monolayer was in a liquid-condensed state, whereas the PL

565

monolayer was at the liquid-condensed/liquid-expanded state boundary. Flipping of

566

molecules between the two leaflets of the asymmetric bilayer once the bilayer is formed

567

would thus be highly improbable. In fact, flipping could only occur during the LS deposition

568

of the LPS leaflet over the PL one, as previously observed for PL/PL bilayers.25 This

569

phenomenon is also promoted by the presence of defects in the PL leaflet. Finally, we also

570

know that a significant PL proportion exists in the outer LPS-rich leaflet of the OM 26, 57. The

571

fact that we found around 25% of dry PL in the outer leaflet can be considered as a relevant

572

model of the OM appropriate for the interaction with plasticins.

573 574

NR and AFM were also used to characterize the interaction of two peptides with the PL/LPS

575

bilayer. They revealed that the neutral plasticin PTCDA1 above its cac only adsorbed

576

transiently to the bilayer and was removed by rinsing without inducing any damage.

577

Conversely, PTCDA1-KF dramatically altered the bilayer structure and its effect was

578

observed even at 0.1 µM, a concentration lower than its cac. In contrast to the results obtained

579

in monolayer studies 38, the peptide was efficient in its monomeric form. However, still 45 to

580

60% of the lipid bilayer remained onto the surface. At 1 µM, the structure of the PL/LPS

581

bilayer was disintegrated by peptide action on the lipid scaffold. Both leaflets of the bilayer

582

were severely damaged by the plasticin that acted through a solubilizing process leading to

583

the complete disruption of the asymmetric bilayer. We cannot tell whether the patches

584

remaining on the surface were pieces of one or the two leaflets of the bilayer or heterogeneous

585

mixtures composed of lipid and peptide. However, the higher phase shift displayed by these

586

features with respect to the mica surface is consistent with heterogeneous material. After

587

rinsing, the bilayer was in all cases washed away from the surface, leaving only fragments and


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Page 28 of 39

588

debris. Force-volume imaging (Fig. S3, Supporting Information) combined to force-distance

589

measurements (Fig. 7E) on areas free of debris demonstrated the absence of organic material

590

beneath the patches which remain on the bare mica surface.

591

At 25 µM PTCDA1-KF, one would expect the complete removal of the bilayer following

592

rinsing. However, NR experiments showed that still some material remained at the surface of

593

the sapphire. As sapphire is a pure oxide, its surface is charged in contact with an aqueous

594

phase at pH above 5. It has been previously shown that strong oxidizing treatment such as

595

piranha or UV/ozone treatment raised the point of zero charge value above 8.56 Attractive

596

electrostatic interactions between the positive charges of the sapphire and the negative PL

597

headgroups might be stronger than those mediated by calcium cations between the PL

598

headgroups and the mica surface. Furthermore, we have observed that the inner leaflet

599

transferred onto mica was much more porous than the one deposited onto sapphire. It is thus

600

possible that the bilayer on mica was more sensitive to the disturbance produced by PTCDA1-

601

KF penetration into the LPS leaflet.

602

As we previously showed with monolayer studies38, upon interaction with the polar face of

603

the LPS leaflet, partial disaggregation occurs for the cationic plasticin, allowing the attractive

604

electrostatic adsorption of oligomers onto the polar face of the LPS leaflet. The natural

605

plasticin remains aggregated upon interaction and unable to penetrate into the LPS monolayer.

606

Both disaggregation and the cationic charge are required to penetrate the upper LPS leaflet.

607

Insertion of the cationic peptide between the lipid molecules led to the disorganization of the

608

LPS monolayer.

609

asymmetric PL/LPS bilayer induced by the cationic plasticin takes place at low peptide

610

concentration (0.1 µM, just below its cac) confirming that the cationic plasticin is also active

611

in the monomeric form, (ii) lipid molecules are extracted out of the bilayer by the interaction

612

with peptide oligomers, creating peptide – lipid complexes that “micellize” the asymmetric

38

The present study demonstrates that: (i) the removal process of the


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Langmuir

613

bilayer components, (iii) these complexes further leave the surface towards the bulk and are

614

flushed by rinsing step. Such a removal process induced by the cationic plasticin PTCDA1-

615

KF is therefore consistent with the carpet scenario5. Kinetic measurements using QCM-D

616

involving one or the other leaflet of the bilayer formed on the sensor could bring useful

617

information about the real-time interaction kinetics and deepen our understanding of the

618

mechanism of action between these model systems and molecules acting at the membrane

619

level.

620 621

5. Conclusion

622

The combination of NR and AFM allowed characterization of the composition and surface

623

morphology of asymmetric PL/LPS bilayers formed on different surfaces. It provided a global

624

understanding of the bilayer structure and lipid organization before and following interaction

625

with two peptides. It highlighted the influence of the substrate on the interaction of the

626

peptides with the supported bilayers. A clear discrimination could be made between the

627

inefficient PTCDA1 and the cationic PTCDA1-KF. At concentrations below its cac, the latter

628

was able to significantly alter the lipid bilayer, and showed a solubilizing effect at higher

629

concentrations. This study confirms the carpeting mechanism for PTCDA1-KF that was

630

deduced from previous monolayer experiments.

631 632

Supporting Information. The Supporting Information is available free of charge on the ACS

633

publication website at DOI.

634 635

Acknowledgments

636

The authors acknowledge the financial support of University Paris-Sud through their

637

“Attractivité 2011” grant program, the Région Ile-de-France (“Equipement mi-lourd 2012”


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638

program, DIM Malinf), the JPK Company for their active support, the NMI3 program for

639

postdoc funding, and the Partnership for Soft condensed Matter at the ILL for the access to

640

the NIMA troughs and the ILL for beamtime allocation. They are also grateful to G. Fragneto

641

(ILL) for NR experiments, M. Aumont-Niçaise (IBBMC, Univ Paris Sud) for DSC

642

measurements.

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Disruption of Asymmetric Lipid Bilayer Models Mimicking the Outer

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