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

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 applications. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology.

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Acknowledgements

The authors would like to thank the Slovenian Research Agency for support (Program grants “Molecular Interactions” P1-0391 and “Biochemical and biophysicochemical characterization of natural substances” P4-0121). We also thank Prof. Sligar’s laboratory for providing us plasmids for membrane scaffold protein expression.

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Correspondence to Gregor Anderluh.

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Rezelj, S., Kozorog, M., Švigelj, T. et al. Cholesterol Enriched Archaeosomes as a Molecular System for Studying Interactions of Cholesterol-Dependent Cytolysins with Membranes. J Membrane Biol 251, 491–505 (2018). https://doi.org/10.1007/s00232-018-0018-y

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Keywords

  • Archaeal lipids
  • Archaeosomes
  • Listeriolysin O
  • Perfringolysin O
  • Cholesterol-dependent cytolysins