Extracellular vesicles (EV) are important factors in characterization of cell condition. In this master thesis we wanted to discover whether the number of large extracellular vesicles (lEV) and/or small extracellular vesicles (sZV) changes after exposing cells to pore forming proteins (PFP) listeriolysin O (LLO) and its two mutants: LLOY406A and LLOA318C-L334C. There was already shown that treating cells with some PFP can lead to higher excretion of EV, but not jet for LLO. Activity of LLO depends on the protein concentration, membrane composition, pH and temperature. In this research project we focused on the effect of wild type LLO and its two mutants LLOY406A and LLOA318C-L334C on cell lines K-562, Jurkat and Caco-2. Previous studies have shown that LLOY406A works similarly as LLO at acidic pH, but at physiological pH it is inactive. LLOA318C-L334C is, when oxidized, locked in such a conformation that pore formation cannot be completed, while in the reduced state it acts similarly as LLO. We characterized response of the cells treated with selected proteins through observing viability and vesiculation using flow cytometry, dynamic light scattering, nanoparticle tracking analysis, and light and confocal microscopy. We found that the cell survival is the lowest after treating cells with LLO at pH 6, followed by LLO at pH 7,4 and LLOY406A at pH 6. LLOA318C-L334C ox can also effect the viability of the cells, but in a smaller scale, and LLOY406A is not litic for the cells at pH 7,4. After treating cells with lytic concentrations of LLO and LLOY406A cells heavily vesiculated and produced more lEV and sEV in comparison to control. At concentrations that were 100-fold lower than lytic ones, cells usually produced less lEV and sEV than control. Cell response to oxidized LLOA318CL334C regarding vesiculation is similar to controls. In future, these findings could helpful at determining cell response to PFP for potential use in the farmaceutical industry.
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