A streamlined method to obtain biologically active TcdA and TcdB toxins fromClostridioides difficile

Abstract

AbstractThe major virulence factors ofClostridioides difficile(C. difficile) are enterotoxin A (TcdA) and cytotoxin B (TcdB). The study of toxins is a crucial step in exploring the virulence of this pathogen. Currently, the toxin purification process is either laborious and time-consuming inC. difficileor performed in heterologous hosts. Therefore, we propose a streamlined method to obtain functional toxins inC. difficile. TwoC. difficilestrains were generated each harboring a sequence encoding a His-tag at the 3’ end ofC. difficile630Δerm tcdAortcdBgenes. Each toxin gene is expressed using the Ptetpromoter inducible by anhydro-tetracycline. The purification yields were estimated to be 0.28 mg per liter and 0.1 mg per liter for rTcdA and rTcdB, respectively. In this study, we successfully developed a simple routine method that allows the production and purification of biologically rTcdA and rTcdB active toxins with similar activities compared to native toxins.