MUTAGENESIS OF THE CLOSTRIDIUM-DIFFICILE TOXIN-B GENE AND EFFECT ON CYTOTOXIC ACTIVITY

Toxins A and B of Clostridium difficile are large cytotoxic proteins t hat share several unusual structural features, including four conserve d cysteines, a potential nucleotide binding site, a hydrophobic region , and a series of contiguous repeating units at the carboxyl terminus. In the following study, we developed a series of toxin B mutants with altered properties in each of these features and examined the effect of the mutation on cytotoxic activity. Altering conserved cysteines to serine resulted in a 90% reduction in activity, whereas altering a hi stidine residue located in the potential nucleotide binding site to gl utamine resulted in a 99% reduction. Removing the repeating units lowe red the activity by 90% whereas removing the repeating units plus a co nserved cysteine located just upstream of the units reduced the activi ty by more than five logs, resulting in an inactive toxin. Deleting th e internal hydrophobic region had a similar effect. Our findings demon strate that these conserved features appear to be important for expres sion of cytotoxic activity.