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Eukaryotic cell-free systems: A novel platform technology for production and functional characterization of pore-forming toxins

: Ramm, F.; Dondapati, S.K.; Zemella, A.; Thoring, L.; Stech, M.; Kubick, S.

Naunyn-Schmiedebergs archives of pharmacology 392 (2019), Supplement 1, pp.S78
ISSN: 0028-1298
ISSN: 1432-1912
German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT Annual Meeting) <85, 2019, Stuttgart>
Association of the Clinical Pharmacology Germany (VKliPha Annual Meeting) <21, 2019, Stuttgart>
Fraunhofer IZI ()

Question: Bacterial infections still cause tremendous health risks worldwide. A variety of pathogenic bacteria in particular causes virulence by pore-forming toxins that insert themselves into membranes and cause cell lysis. Since highly resistant bacterial strains have evolved over the last decades, new methods for the characterization of bacterial toxins are needed in order to develop new and specific diagnostic tools as well as to improve present-day treatment regimens.
Methods: In vivo production of toxins is rather difficult as toxins harm the cells" viability. Cell-free protein synthesis has emerged as a fast and cost-efficient method to synthesize and characterize a variety of toxins while using a cell lysate rather than viable cells. Here, we describe the cell-free synthesis and functional characterization of enterotoxins from Bacillus cereus as well as Staphylococcus aureus, Cytolysin from Vibrio vulnificus and Aerolysin from Aeromonas hydrophila.Cell-based toxicity assays, blood agar plates and electrophysiological measurements on planar lipid bilayers were used to determine the toxins" functionality.
Results: In our studies, we showed the successful synthesis and characterization of various pore-forming toxins and the ability to use the cell-free system in versatile ways.
Conclusion: This platform technology enables a fast and efficient characterization of a variety of toxins as a prerequisite to develop diagnostic methods and treatments.