Comparison of wild type with recombinant Clostridium difficile toxin A.
Toxins A and B from Clostridium difficile are single-chain proteins of 308,000 and 270,000 Da, respectively. They possess transferase activity to monoglucosylate proteins of the Rho GTPase family whereby Rho, Rac, and Cdc42 are the canonical substrates. For application of these toxins as specific Rho GTPase inhibitors the highest possible purity is of crucial interest. We, therefore, expressed recombinant His-tagged toxin A using the Bacillus megaterium expression system. Specific antisera raised against the native toxin A from C. difficile and the recombinant toxin, respectively, showed identical sensitivity and specificity in Western blot and ELISA analyses towards both toxins. By comparison of both toxins in functional studies we showed that the recombinant toxin was about two times more cytotoxic than the native toxin, and the glucosyltransferase-activity of the recombinant toxin was even 10-fold increased. However, recombinant toxin A showed one essential difference to the classically purified one. The reported transferase-independent effect of toxin A to release cytochrome c from isolated mitochondria was not exhibited by the recombinant toxin A. This putative mitochondrial effect decreased with increased purity of toxin A, and was absent with recombinant toxin, strongly suggesting an clostridial contamination responsible. In summary, we tested the recombinant toxin A to be at least an adequate substitute for the native toxin, bearing the advantage of a rapid single-step purification and the absence of biological active contaminations.
Gerhard
Ralf
R
Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. gerhard.ralf@mh-hannover.de
Burger
Silke
S
Tatge
Helma
H
Genth
Harald
H
Just
Ingo
I
Hofmann
Fred
F
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity*; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000201:enzymology*; D045304:Cytochromes c / Q000032:analysis; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity*; D004797:Enzyme-Linked Immunosorbent Assay; D005964:Glucosyltransferases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000187:drug effects; D041681:NIH 3T3 Cells; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000633:toxicity
D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D004768:Enterotoxins; D011994:Recombinant Proteins; C041348:tcdA protein, Clostridium difficile; D045304:Cytochromes c; D005964:Glucosyltransferases