Browsing by Author "Clifton, LA"

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    Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic
    (The Royal Society, 2013-10-16) Clifton, LA; Skoda, MWA; Daulton, E; Hughes, AV; Le Brun, AP; Lakey, JH; Holt, SA
    The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions. © 2013, The Royal Society.
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    Effect of divalent cation removal on the structure of gram-negative bacterial outer membrane models
    (American Chemical Society, 2015-01-13) Clifton, LA; Skoda, MWA; Le Brun, AP; Ciesielski, F; Kuzmenko, I; Holt, SA; Lakey, JH
    The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration. © 2014 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
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    Examining the creation and destruction of model bacterial surfaces
    (International Conference on Neutron Scattering, 2017-07-12) Clifton, LA; Skoda, MWA; Hughes, A; Holt, SA; Lakey, J
    Bacteria are differentiated into two main groups, Gram-positive or Gram-negative, based on the Gram stain which detects the thick peptidoglycan cell wall of gram positive bacteria.Gram-negative bacteria are of particular biomedical and technological interest due to their role in disease, the increasing antibiotic resistance if some species and their utility in many biotechnological processes. The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS is a polyanionic molecule, with numerous phosphate groups present in the Lipid A and core oligosaccharide regions. We have attempted to create GNB-OM assays which are amenable to molecular level characterisation. These systems are asymmetric phospholipid : lipopolysaccharide membranes deposited at the solid/liquid interface and consist of either solid supported bilayers at the silicon/water interface or floating supported bilayers at the gold/water interface. The analysis of these membrane models by neutron reflectometry has provided new insights into the OM and interactions with it. Examples of this include providing conformation of the activity of anti-bacterial proteins, the role of the lipopolysaccharide polysaccharide chains in protecting the bacterium and the importance of divalent cations in stabilising the OM structure.
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    Investigating the interactions of the 18 kDa translocator protein and its ligand PK11195 in planar lipid bilayers
    (Elsevier, 2014-03) Hatty, CR; Le Brun, AP; Lake, V; Clifton, LA; Liu, GJ; James, M; Banati, RB
    The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18 kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~ 10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51 Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug–membrane interactions may contribute to the apparent affinity of TSPO ligands. © 2013, Elsevier B.V.
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    Structural characterization of a model gram-negative bacterial surface using lipopolysaccharides from rough strains of escherichia coli
    (American Chemical Society, 2013-06-01) Le Brun, AP; Clifton, LA; Halbert, CE; Lin, B; Meron, M; Holden, PJ; Lakey, JH; Holt, SA
    Lipopolysaccharides (LPS) make up approximately 75% of the Gram-negative bacterial outer membrane (OM) surface, but because of the complexity of the molecule, there are very few model OMs that include LPS. The LPS molecule consists of lipid A, which anchors the LPS within the OM, a core polysaccharide region, and a variable O-antigen polysaccharide chain. In this work we used RcLPS (consisting of lipid A plus the first seven sugars of the core polysaccharide) from a rough strain of Escherichia coli to form stable monolayers of LPS at the air?liquid interface. The vertical structure RcLPS monolayers were characterized using neutron and X-ray reflectometry, while the lateral structure was investigated using grazing incidence X-ray diffraction and Brewster angle microscopy. It was found that RcLPS monolayers at surface pressures of 20 mN m(-1) and above are resolved as hydrocarbon tails, an inner headgroup, and an outer headgroup of polysaccharide with increasing solvation from tails to outer headgroups. The lateral organization of the hydrocarbon lipid chains displays an oblique hexagonal unit cell at all surface pressures, with only the chain tilt angle changing with surface pressure. This is in contrast to lipid A, which displays hexagonal or, above 20 mN m(-1), distorted hexagonal packing. This work provides the first complete structural analysis of a realistic E. coli OM surface model. © 2013, American Chemical Society.