The ability of pore-forming proteins to interact with various analytes has found vast applicability in single molecule sensing and characterization. In spite of their abundance in organisms from all kingdoms of life, only a few pore-forming proteins have been successfully reconstituted in artificial membrane systems for sensing purposes. Lysenin, a pore-forming toxin extracted from the earthworm E. fetida, inserts large conductance nanopores in lipid membranes containing sphingomyelin. Here we show that single lysenin channels may function as stochastic nanosensors by allowing the short cationic peptide angiotensin II to be electrophoretically driven through the conducting pathway. Long-term translocation experiments performed using large populations of lysenin channels allowed unequivocal identification of the unmodified analyte by Liquid Chromatography-Mass Spectrometry. However, application of reverse voltages or irreversible blockage of the macroscopic conductance of lysenin channels by chitosan addition prevented analyte translocation. This investigation demonstrates that lysenin channels have the potential to function as nano-sensing devices capable of single peptide molecule identification and characterization, which may be further extended to other macromolecular analytes.

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.

No abstract

Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations might interfere with host-cell-signaling pathways, causing apoptosis. In the present investigation we demonstrate that a relatively low concentration of HlyA induces morphological changes and phosphatidylserine (PS) externalization of human erythrocytes. On the other hand, the unacylated nonhemolytic form of HlyA, ProHlyA induces similar morphological changes but no PS externalization. We performed osmoscan experiments to test the effect of both proteins on erythrocytes structure. HlyA treated erythrocytes show increased membrane fragility and cell volume as well as diminished cytoplasmic viscosity and S/V ration. ProHlyA-treated erythrocyte are not different from control ones. Since PS exposure of erythrocytes is known to induce cell adhesion, we used a dynamic cell adhesion platform to study the consequences of HlyA vs ProHlya exposure of erythrocytes on their adhesion to human endothelial cells (HMEC). Results indicate that HlyA-treated erythrocytes adhere more to endothelial cells than Pro-treated erythrocytes at low flux (0.5 din). At higher fluxes (1 and 2 din), however, HlyA-treated erythrocytes detached easily than control ones, indicating that the adherence is weak. We also study the efflux of ATP from erythrocytes treated with both toxins by luciferinluciferase luminescence. Results demonstrate that HlyA induces the efflux of ATP while ProHlyA does not. Since PS exposure was suggested to simultaneously increase extracellular ATP and adhesion to the vascular endothelium, and erythrocyte derived ATPe can alter the caliber of the vascular lumen, future experiments will be designed to relate HlyA induced efflux of ATP of erythrocytes with their adhesion and interaction with endothelial cells.

Bacteriophage T4 is one of the most common and complex tailed viruses from the family Myoviridae, and it infects E. coli using a highly efficient contractile genome delivery machine. Phage T4 possesses a multi-protein capsid containing the genomic DNA and a long contractile tail assembly that consists of a rigid tail tube surrounded by a six helical-stranded sheath. The tail assembly transmits the genomic DNA from the capsid to the host during injection. While the atomistic structure of phage T4 has been studied extensively using Cryo-EM and X-Ray crystallography, the dynamics of the injection process is not well understood. Questions remain regarding the energetics of injection as well as the pathway and time scale of the large conformational change of the sheath. In this study, we employ a dynamic multi-scale model to address these fundamental questions. To this end, a two-stage solution process is employed; first, a molecular dynamics (MD) simulation is carried out to estimate the elastic properties of the sheath strands. Second, a continuum model is developed for the entire-assembled T4 injection machinery using the elastic constants estimated from MD. The resulting multi-scale model simulates the dynamics of the sheath represented by six interacting helical strands that are coupled to the capsid represented by a massive cylinder. Dynamic simulations reveal the time scale, the pathway of sheath contraction, and the energetics driving the injection process. While these results are specific to T4 as an example, the resulting model and methodology may also inform the future design of nanotechnology injection devices.