Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

Abstract

Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin’s transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel’s conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.

Lysenin Channels as Sensors for Ions and Molecules

“…Irreversible channel blockage by cationic polymers can be realized at concentrations in the nM range [29]. Given the bio-inertness of chitosan, this particular irreversible blockage was recently exploited for temporary permeabilization of live cells and access of non-permeant molecules to the cytosol while maintaining an excellent viability of the target cells [93]. Reversible permeabilization of artificial spherical cell membranes (liposomes) was achieved by employing lysenin channels, La 3+ ions, and EDTA [93], which may open novel avenues for drug loading into liposomal carriers and controlled release at the desired sites.…”

Non-viral transfection technologies for next-generation therapeutic T cell engineering

“…Following a rather timeconsuming permeabilization process of 6 h, mean pDNA transfection efficiencies of 15% could be achieved in PBMCs (Nateri et al, 2005). Permeabilization of the T cell membrane can also be obtained by bacterial pore-forming toxins, such as Streptolysin-O (SLO) (Alexander et al, 1989;Kobayashi et al, 2020) or Lysenin (Shrestha et al, 2020), although the applicability of these toxins for T cell engineering remains largely unexplored. This is not surprising, knowing that the generated pore sizes are limited to delivery of macromolecules <150 kDa (Walev et al, 2001), which makes it incompatible with most of the effector molecule types for T cell engineering (Stewart et al, 2018).…”

Rapid Production and Purification of Dye-Loaded Liposomes by Electrodialysis-Driven Depletion

“…For our investigations we used the prototype pore-forming toxin lysenin, which introduces large-conductance pores in artificial and natural membranes containing sphingomyelin [56][57][58][59]. However, for relevancy with regards to the membrane thickness, one may assume that lysenin may not span multiple bilayers [60], therefore the changes in membrane permeability are specific to unilamellar liposomes. Liposomes consisting of Aso, SM, and Chol (10:4:4 weight ratio) were produced and loaded with AO by electrodialysis as described in the previous sections and analyzed by fluorescence spectroscopy.…”