Abstract
BackgroundListeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes and facilitates the intracellular survival of the pathogen. Some of its characteristics endorse the growing popularity of LLO for use in biotechnology, particularly in the development of novel vaccines. Here, we evaluate the use of LLO to eradicate leukaemia cells.ResultsA purified LLO preparation was obtained by affinity chromatography. The LLO preparation procedure was optimized and purified LLO was tested for optimal conditions of storage including temperature, application of proteinase inhibitors and serum components. We demonstrated the possibility of regulating LLO activity by adjusting cell membrane cholesterol content. The LLO preparation had haemolytic activity and had a cytotoxic effect on the human T-leukaemia Jurkat cell line as well as mouse and human peripheral blood mononuclear cells.ConclusionsLLO has a very potent cytotoxic activity towards human leukocytes. Importantly, the cytotoxic activity was easily regulated in vitro and could be restricted to areas containing malignant cells, raising the possibility of future clinical application of LLO for leukaemia treatment.
Highlights
Listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes and facilitates the intracellular survival of the pathogen
Purification of Listeriolysin O The synthesis and affinity purification of His-tagged listeriolysin O (LLO) from E. coli harbouring a pET29b-hly plasmid was optimized with a set of buffers with the following gradient: buffer pH ranging from 5 to 8 and NaCl concentration from 0 to 0.5 M
The use of low pH buffer offers two advantages over commonly used pH 8 elution buffer: (i) it effectively reduces the ability of nickel - nitrilotriacetic acid (Ni-NTA) resin to bind proteins, facilitating the elution of a higher protein yield with lower imidazole concentrations, and (ii) it preserves the activity of the LLO protein
Summary
Listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes and facilitates the intracellular survival of the pathogen. Most bacterial pathogens produce toxins, which are important virulence factors. Some toxins act on cytoplasmic membranes, whereas others are receptor-targeted toxins or membrane damaging toxins. The latter toxins are referred to as cytolysins, and are produced by numerous Gram-positive and Gram-negative bacteria. Some of these toxins display enzymatic activity, while others have cytolytic capacity without enzymatic activity. For example some phospholipases facilitate the action of pore-forming cytolysins by hydrolysing membrane lipids [1]
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