Curved membrane structures induced by native lipids in giant vesicles

Nair, Karthika S.; Raj, Neethu B; Nampoothiri, K. Madhavan; Mohanan, Gayathri; Acosta‐Gutiérrez, Silvia; Bajaj, Harsha

doi:10.1016/j.jcis.2021.12.098

Cited by 9 publications

(18 citation statements)

References 47 publications

(78 reference statements)

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“…The arrangement of LPS reconstitution in OM vesicles is investigated as described by a fluorescence quenching assay. ,, OM vesicles fluorescently labeled with 0.25 or 10 mol % Alexa Fluor 488-conjugated LPS are subjected to incubation of a quencher dye trypan blue (500 μM) for 5 min followed by imaging. Subsequently, 500 μM or 1 mM SDS is added to the vesicles and incubated for 5 min to rupture the membrane, expose the inner leaflet to trypan blue, and finally imaged.…”

Section: Methodsmentioning

confidence: 99%

“…Vesicles are prepared by polyvinyl alcohol (5% wt/vl PVA) gel-assisted swelling method as described. − E. coli polar lipid extract [referred to as inner-membrane (IM) lipid] is utilized to construct IM vesicles or LPS-free vesicles mimicking the bacterial inner-membrane composition using a PVA-based gel-assisted method.…”

Section: Methodsmentioning

confidence: 99%

“…Effective LPS incorporation in OM vesicles at pH = 5 and 7 is quantified using fluorescence imaging . Vesicles are prepared at different mol % of LPS to IM lipids (8:48, 6:48, 4:48, 2:48, 1:48).…”

Section: Methodsmentioning

confidence: 99%

“…32−34 Recently, our group constructed GUVs with LPS to investigate the role of bacterial lipids, including LPS, in the membrane bending and transformation process. 35 Here, we advance our work into engineering outer membrane (OM)-mimicking giant vesicle models at various pH conditions. Notably, the effective LPS reconstituted and its arrangement in giant vesicles is determined.…”

Section: ■ Introductionmentioning

confidence: 99%

“…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: Methodsmentioning

confidence: 99%