Cytopathogenic effect of Streptococcus pneumoniae native pneumolysin in CHO-K1 cells

Background. Streptococcus pneumoniae (S. pneumoniae, pneumococci) is an opportunistic bacterium that causes inflammatory diseases in humans. One of the virulence factors of S. pneumoniae is pneumolysin (Ply), a cholesterol-dependent hemolytic toxin that interacts with cholesterol in eukaryotic cell membranes, forms pores and leads to cell destruction and death.

Aim. Immunochemical characteristics of pneumolysin and evaluation of its cytotoxic effect in the culture of Chinese hamster ovarian cells CHO-K1.

Materials and methods. To obtain Ply, the S. pneumoniae serotype 3 strain was cultivated in brain heart broth, bacterial cells were pelleted by centrifugation and subjected to ultrasonic disintegration. The presence of pneumolysin in the resulting preparation was confirmed by immunoblotting with monoclonal antibodies to recombinant pneumolysin. The cytopathogenic effect of Ply was studied in a culture of proline-dependent epithelial-like Chinese hamster ovary cells CHO-K1 in RPMI-1640 medium. The hemolytic activity of Ply was assessed in a reaction with mouse erythrocytes.

Results. Using ultrasonic disintegration of cells of the S. pneumoniae serotype 3 strain followed by precipitation with ammonium sulfate, a protein that formed a band on electrophoresis at a level corresponding to the molecular weight of Ply (53 kDa) was obtained. The protein possessed the ability to lyse mouse erythrocytes. The authenticity of Ply was confirmed by immunoblotting with monoclonal antibodies to recombinant pneumolysin. Ply had a cytopathogenic effect on Chinese hamster ovary CHO-K1 cells. The minimum dose of the protein that induced a toxic effect on cells, manifested in the appearance of round and small cells along with normal cells, was 16.4 μg/ml per protein. When the concentration was increased up to 65.6 μg/ml, only small round cells were detected. Further increase in concentration resulted in complete destruction of CHO-K1 cells.

Conclusion. Pneumolysin can be used to develop new drugs for the treatment of pneumococcal infections.

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