Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides

Stephan, Mareike; Dunsing, Valentin; Pramanik, Shreya; Chiantia, Salvatore; Barbirz, Stefanie; Robinson, Tom; Dimova, Rumiana

doi:10.1016/j.bpj.2022.12.017

Cited by 9 publications

(6 citation statements)

References 78 publications

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“…Several studies have reported asymmetric arrangement of LPS in GUVs using the water-in-oil method in bulk or microfluidic-based chip. 31−34 Wild-type LPS has been reconstituted in the outer leaflet of GUVs, where heterogeneous LPS reconstitution (1−16 mol %) is observed in a vesicle population 34 similar to the LPS heterogeneity noted here (Figure 2A,B). Complete LPS asymmetry is not feasible using the hydration method employed here.…”

Section: ■ Results and Discussionsupporting

confidence: 70%

“…Indeed, asymmetric reconstitution of LPS in GUVs has been achieved via water-in-oil emulsion preparation methods in bulk or microfluidic chips. Such studies primarily used truncated LPS or full-length LPS with mixtures of lipids as the inner leaflet of vesicles remotely mimicking physiological bacterial membranes. − Recently, our group constructed GUVs with LPS to investigate the role of bacterial lipids, including LPS, in the membrane bending and transformation process …”

Section: Introductionmentioning

confidence: 99%

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Bacterial Outer-Membrane-Mimicking Giant Unilamellar Vesicle Model for Detecting Antimicrobial Permeability

2023

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The construction of bacterial outer membrane models with native lipids like lipopolysaccharide (LPS) is a barrier to understanding antimicrobial permeability at the membrane interface. Here, we engineer bacterial outer membrane (OM)mimicking giant unilamellar vesicles (GUVs) by constituting LPS under different pH conditions and assembled GUVs with controlled dimensions. We quantify the LPS reconstituted in GUV membranes and reveal their arrangement in the leaflets of the vesicles. Importantly, we demonstrate the applications of OM vesicles by exploring antimicrobial permeability activity across membranes. Model peptides, melittin and magainin-2, are examined where both peptides exhibit lower membrane activity in OM vesicles than vesicles devoid of LPS. Our findings reveal the mode of action of antimicrobial peptides in bacterial-membrane-mimicking models. Notably, the critical peptide concentration required to elicit activity on model membranes correlates with the cell inhibitory concentrations that revalidate our models closely mimic bacterial membranes. In conclusion, we provide an OM-mimicking model capable of quantifying antimicrobial permeability across membranes.

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Section: ■ Results and Discussionsupporting

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Bacterial Outer-Membrane-Mimicking Giant Unilamellar Vesicle Model for Detecting Antimicrobial Permeability

2023

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“…These molecular curvatures, are not to be confused with the curvature of the membrane, which in our system is relatively low and of the order of 0.1 m -1 . High membrane curvature is typically generated by asymmetry of the bilayer leaflet composition [53][54][55][56] or the solution across the membrane [35,57], whereby the latter was also shown to modulate the phase state of charged membranes [58][59][60]. We noticed that the relative content of POPC and POPE had a strong influence on the size of the formed GUVs.…”

Section: Popc/pope Guvs As Basic Models Of the Inner Cell Membrane Of...mentioning

confidence: 85%

“…The small size of GUVs can limit their application in studying the diffusion of lipids and incorporated molecules. Methods such as fluorescence recovery after photobleaching, which is based on the bleaching of a small spot, and observation of its recovery, will not be applicable in determining diffusion coefficient as the standard size of the spot (typically a few micrometers in diameter) is usually comparable with the average size of the GUVs obtained here (~5-10 μm); an approach based on half-vesicle bleaching might still be applicable [54]. Nevertheless, the average size of the bacterial cell lies between 1-2 μm, with only a few exceptions such as e.g.…”

Section: Resultsmentioning

confidence: 99%

Tunable biomimetic bacterial membranes from binary and ternary lipid mixtures and their application in antimicrobial testing

Krok

Stephan

Dimova

et al. 2023

Preprint

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Reconstruction of accurate yet simplified mimetic models of cell membranes is a very challenging goal of synthetic biology. To date, most of the research focuses on the development of eukaryotic cell membranes, while reconstitution of their prokaryotic counterparts has not been fully addressed, and the proposed models do not reflect well the complexity of bacterial cell envelopes. Here, we describe the reconstitution of biomimetic bacterial membranes with an increasing level of complexity, developed from binary and ternary lipid mixtures. Giant unilamellar vesicles composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); PC and phosphatidylglycerol (PG); PE and PG; PE, PG and cardiolipin (CA) at varying molar ratios were successfully prepared by the electroformation method. Each of the proposed mimetic models focuses on reproducing specific membrane features such as membrane charge, curvature, leaflets asymmetry, or the presence of phase separation. GUVs were characterized in terms of size distribution, surface charge, and lateral organization. Finally, the developed models were tested against the lipopeptide antibiotic daptomycin. The obtained results showed a clear dependency of daptomycin binding efficiency on the amount of negatively charged lipid species present in the membrane. We anticipate that the models proposed here can be applied not only in antimicrobial testing but also serve as platforms for studying fundamental biological processes in bacteria as well as their interaction with physiologically relevant biomolecules.

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“…Both asymmetric and symmetric vesicles were obtained using the inverted emulsion method (also known as the phase-transfer method) [41][42][43] with some modifications. This method requires dissolving the lipids in oil.…”

Section: Vesicle Preparationmentioning

confidence: 99%

Effect of leaflet asymmetry on the stretching elasticity of lipid bilayers with phosphatidic acid

Drabik

Hinc

Stephan

et al. 2022

Preprint

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Asymmetry in membranes strongly affects their biophysical characteristics and behaviour. It is a very important factor to consider for understanding the functioning of membranes in biological systems, especially in regards to lipid molecules that are known to localize in one leaflet over the other. Phosphatidic acid is one representative of this family of molecules. Reported to play a crucial role in lipid metabolism and suspected to participate in cellular signalling, its specific nature might only be exhibited in asymmetric systems. Here, using giant unilamellar vesicles, we studied two asymmetric phosphocholine-based bilayer systems with 1-palmitoyl-2-oleoyl phosphatidic acid (POPA) localized in either the inner or the outer membrane leaflet. We systematically characterized and compared the investigated systems to their symmetric counterpart. To verify and quantify the presence of POPA in the membrane, α-synuclein-mEGFP was used. We report an increase in the area compressibility and bending rigidity moduli in the asymmetric membranes compared to the symmetric ones. Furthermore, we show area compressibility differences depending on the localization of POPA in the leaflets. The results suggest the asymmetry affects both mechanics and phase state of the membrane.

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Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides

Cited by 9 publications

References 78 publications

Bacterial Outer-Membrane-Mimicking Giant Unilamellar Vesicle Model for Detecting Antimicrobial Permeability

Bacterial Outer-Membrane-Mimicking Giant Unilamellar Vesicle Model for Detecting Antimicrobial Permeability

Tunable biomimetic bacterial membranes from binary and ternary lipid mixtures and their application in antimicrobial testing

Effect of leaflet asymmetry on the stretching elasticity of lipid bilayers with phosphatidic acid

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