Cholesterol Enriched Archaeosomes as a Molecular System for Studying Interactions of Cholesterol-Dependent Cytolysins with Membranes

The Journal of Membrane Biology
Saša RezeljGregor Anderluh

Abstract

Archaeosomes are vesicles made of lipids from archaea. They possess many unique features in comparison to other lipid systems, with their high stability being the most prominent one, making them a promising system for biotechnological applications. Here, we report a preparation protocol of large unilamellar vesicles, giant unilamellar vesicles (GUVs), and nanodiscs from archaeal lipids with incorporated cholesterol. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. Surface plasmon resonance, sedimentation assays, intrinsic tryptophan fluorescence measurements, calcein release experiments, and GUVs experiments showed that members of cholesterol-dependent cytolysins, listeriolysin O (LLO), and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, we observed specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. GUVs made from archaeal lipids were highly stable, which could be beneficial for synthetic biology application...Continue Reading

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Citations

Jul 1, 2018·The Journal of Membrane Biology·Ana-Nicoleta Bondar, Sandro Keller
Mar 17, 2021·Biochimica Et Biophysica Acta. Biomembranes·Nejc PetrišičGregor Anderluh

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Methods Mentioned

BETA
in vitro transcription
gel filtration
adsorption chromatography
electrophoresis
chip
Fluorescence
size-exclusion chromatography
dynamic light scattering
fluorescence microscopy

Software Mentioned

ImageJ
Digital Micrograph software
Malvern ZetaSizer

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