Study on Expression and Cytotoxicity of Recombinant Clostridium difficile Cytotoxin (Toxin B)

Abstract

Clostridium difficile, an anaerobic, gram-positive bacillus, causes several diseases, including diarrhea, colitis, and septicemia, resulting in death. C. difficile-associated diseases have increased significantly due to the emergence of a highly virulent strain of C. difficile. Nosocomial infections, particularly, have serious repercussions. Toxin B is essential for C. difficile virulence. Here, to elucidate the mechanism underlying C. difficile virulence due to toxin B, we constructed a recombinant partial toxin B and analyzed its cytotoxic activity. The PCR-amplified toxin B fragment from C. difficile GAI 99093 was recombined with the pET101/D-TOPO vector. Recombinant protein expression was then induced with isopropyl-β-D(-)-thiogalactopyranoside. The recombinant protein was refolded to yield a purified active form. In cytotoxic assay, vero cells were grown to confluence in 96-well plates and various concentrations of refolded protein were added to each well. Culture medium devoid of toxins A and B was used for the negative control. After incubation at
37°C/24 h in 5% CO2, the morphological changes were observed by microscopy. The protein was then expressed in Escherichia coli, and its relative molecular weight was predicted size (24 kDa) of the recombinant protein. Vero cell cytotoxicity assays were conducted with the refolded recombinant protein. The cytotoxic activity observed was toxin B mediated.