Listeriolysin O (LLO) is a pore-forming protein of the bacteria Listeria monocytogenes. Its activity depends on pH. At the Institute of Chemistry, an LLO Y406A mutant was prepared. Its activity at room temperature is more dependent on pH than the activity of its wild-type (LLO wt). LLO Y406A is therefore interesting for biotechnological applications. The purpose of our study was to define the effects of LLO wt and LLO Y406A on blood cells at a physiological temperature (37 °C). We compared the response of different cancer blood cells (K-562, Jurkat, Raji) with the response of healthy cells isolated from blood. Cell condition was determined through dying, tracking secreted extracellular vesicles, observing the formation of reactive oxygen species (ROS) and the amount of ATP. LLO Y406A toxicity to blood cells, at physiological pH (7.4), was found to be very low. Cell treatment with LLO Y406A resulted in 20% cell mortality at 300–7000 times higher concentrations than with LLO. In addition, we found that LLO wt and LLO Y406A are more toxic to cancerous blood cells than to healthy blood cells in physiological conditions. LLO wt treatment resulted in 20% mortality of healthy cells at 1.7–300 times higher concentrations than of cancer cells, and LLO Y406A at 3–30 times higher concentrations in healthy cells than in cancer cells. By lowering pH, the activity of LLO Y406A further increases. At pH 6.5, it causes 20% mortality of healthy cells at 4–40 times higher concentrations than of cancer cells. Increased vesiculation in response to LLO wt or LLO Y406A was detected at 10–100 times lower protein concentration than cell death. LLO wt and LLO Y406A did not cause the increase of ROS, but LLO wt caused an increase in ATP, while LLO Y406A did not. At higher concentrations, we noticed a decrease in ATP.
|
