Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition

Maktabi, Sepehr; Schertzer, Jeffrey W.; Chiarot, Paul R.

doi:10.1039/c9sm00223e

Cited by 22 publications

(30 citation statements)

References 89 publications

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“…By using the phase transfer method to reconstitute asymmetric GUVs, we prepared asymmetric vesicles composed of LPS in the outer and phospholipids in the inner leaflet for the first time. Maktabi et al (2019) published in 2019 a method to produce asymmetric LPS-containing vesicles as water-in-oil-in-water double emulsions prepared by a microfluidics approach. Attempts to reconstitute asymmetric LPS-containing vesicles by cyclodextrin-catalysis ( Doktorova et al, 2018 ) did not work out so far.…”

Section: Discussionmentioning

confidence: 99%

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria

Paulowski

Donoghue

Nehls

et al. 2020

Front. Cell Dev. Biol.

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The architecture of the lipid matrix of the outer membrane of Gram-negative bacteria is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is built up by glycolipids. For most Gram-negative species, these glycolipids are lipopolysaccharides (LPS), for a few species, however, glycosphingolipids. We demonstrate experimental approaches for the reconstitution of these asymmetric membranes as (i) solid supported membranes prepared by the Langmuir-Blodgett technique, (ii) planar lipid bilayers prepared by the Montal-Mueller technique, and (iii) giant unilamellar vesicles (GUVs) prepared by the phase transfer method. The asymmetric GUVs (aGUVs) composed of LPS on one leaflet are shown for the first time. They are characterized with respect to their phase behavior, flipflop of lipids and their usability to investigate the interaction with membrane active peptides or proteins. For the antimicrobial peptide LL-32 and for the bacterial porin OmpF the specificity of the interaction with asymmetric membranes is shown. The three reconstitution systems are compared with respect to their usability to investigate domain formation and interactions with peptides and proteins.

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Section: Discussionmentioning

confidence: 99%

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria

Paulowski

Donoghue

Nehls

et al. 2020

Front. Cell Dev. Biol.

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“…As an alternative to artificial partition systems, giant unilamellar vesicles, GUV, can be used. − Such vesicles can be prepared through lipid extrusion and classified according to their size as small (50–200 nm), large (200 nm to 1 μm), and giant (1–10 μm). Such particles are characterized by reasonably stable electrophysiological conditions and are conducive to the reconstitution of biomimetic membranes.…”

Section: Om Porins and Translocatorsmentioning

confidence: 99%

The Whole Is Bigger than the Sum of Its Parts: Drug Transport in the Context of Two Membranes with Active Efflux

et al. 2021

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Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis–Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux.

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“…Maktabi et al (2019) described a microfluidic technique for fabricating monodisperse asymmetric giant unilamellar vesicles, equipped with Gram-negative's bacterial outer membrane lipid composition. They generated 50-150 μm diameter water-in-oil-in-water double emulsions which simulate the inner and outer leaflets of the lipid bilayer (Maktabi et al, 2019). Extraction processes by ethanol and micropipette aspiration triggered adhesive interaction between the two lipid monolayers assembled on the water-oil and oil-water interfaces (i.e., dewetting transition), forcing them to contact and to form lipid bilayer membranes that resemble Gram negative's bacterial walls.…”

Section: Discussionmentioning

confidence: 99%

Towards Dewetting Monoclonal Antibodies for Therapeutical Purposes

Tozzi¹

2019

Preprint

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Dewetting transition -a concept borrowed from fluid mechanics -is a physiological process which takes place inside the hydrophobic pores of ion channels. This transient phenomenon causes a metastable state which forbids water molecules to cross the microscopic receptors' cavities. This leads to a decrease of conductance, a closure of the hole and, subsequently, severe impairment of cellular performance. We suggest that artificially-provoked dewetting transition in ion channels' hydrophobic pores could stand for a molecular candidate to erase detrimental organisms, such as viruses, bacteria and cancer cells. We describe a novel type of high-affinity monoclonal antibody, which: a) targets specific trans-membrane receptor structures of harmful or redundant cells; b) is equipped with lipophilic and/or hydrophobic fragments that prevent physiological water flows inside ion channels. Therefore, we achieve an artificial dewetting transition inside receptors' cavities which causes transmembrane ionic flows discontinuity, channel blockage and subsequent damage of morbid cells. As an example, we describe dewetting monoclonal antibodies targeting the M2 channel of the Influenza A virus: they might prevent water to enter the pores, thus leading to virion impairment.In fluid mechanics, dewetting is a process occurring at solid-liquid or liquid-liquid interfaces. Dewetting stands for the rupture of the thin, liquid continuous film on the substrate's surface, leading to formation of irregular patterns of droplets. The opposite process is called spreading. Four stages can be recognized as dewetting proceeds (Sharmaa, 1996): (a) film rupture; (b) hole expansion and coalescence to form a polygonal "cellular" pattern. The dry patches augment when the material is gathered in the rim surrounding the growing hole; (c) disintegration of polymer ridges into spherical drops, due to Rayleigh instability. The droplets' size and spacing may vary over several orders of magnitude, since dewetting process starts from randomly formed holes inside the film. A two-tier surface exhibits a two-stage wetting transition: first impalement at microscale texture, then at nanoscale; (d) fingering instability of hole rims during their expansion (Sharmaa, 1996). This process, borrowed by physics, has been recently extended to describe also microscopic biological phenomena. In particular, dewetting transitions may occur inside the hydrophobic pores of cellular ion channels. In the narrowest, more hydrophobic parts of receptors' holes, a metastable state of dewetting transition forbids water molecules to get inside the cavities, leading to decrease in conductance, channel closure and impairment of cellular activities. We show how this peculiar process can be artificially produced to alter the physiological activity of noxious pathogens, such as viruses, bacteria and tumoral cells. In particular, we suggest the manufacture of monoclonal antibodies (against cellular receptors) equipped with lipophilic/hydrophobic caps. In the sequel, we will term these antibod...

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Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition

Abstract: We report on a microfluidic technique for fabricating monodisperse asymmetric giant unilamellar vesicles (GUVs) possessing the Gram-negative bacterial outer membrane lipid composition.

Cited by 22 publications

References 89 publications

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria

The Whole Is Bigger than the Sum of Its Parts: Drug Transport in the Context of Two Membranes with Active Efflux

Towards Dewetting Monoclonal Antibodies for Therapeutical Purposes

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