Mutations in the autoprocessing domain active site and the cleavage site result in TcdB proteins with impaired autoprocessing activity <i>in vitro</i> and in cells.

<p><i>A</i>, Autoprocessing was induced <i>in vitro</i> by incubating wild-type TcdB and TcdB mutants with multiple InsP6 concentrations and 1 mM DTT at 37°C. After 2 hours, the proteins were subjected to SDS-PAGE and visualized with Coomassie stain. A representative series of gels is shown from experiments performed in triplicate. <i>B</i>, Three replicates of the experiments shown in panel <b>A</b> were quantified by densitometry. Bands corresponding to TcdB 544–2366 were quantified and normalized to the band corresponding to TcdB 1–2366 without InsP6. Error bars reflect the standard deviation of the percent cleavage between three experiments. The data indicate that wild-type TcdB autoproteolysis can be detected at concentrations of 1 uM to 1 mM InsP6. By comparison, TcdB mutants C698S, C698A, and H653A were completely inactive for autoprocessing at all InsP6 concentrations. The TcdB D587N and L543A had some residual activity, but autoprocessing activity was impaired relative to wild-type. <i>C</i>, GTDs of autoprocessing mutants are not released in cells. HeLa cells were synchronized for 30 minutes at 4°C, then intoxicated with 10 nM toxin. Intoxicated cells were incubated at 4°C for an hour before being moved to 37°C. Cells were harvested after 50 minutes, and cell lysates were prepared for SDS PAGE and Western blot. The blot was probed with antibodies against the TcdB GTD, unglucosylated Rac1, total Rac1, and GAPDH. While release of the GTD in cells intoxicated with wild-type TcdB was detected, the free GTD was not detected in cells treated with autoprocessing deficient mutants. The absence of signal with an antibody that recognizes unglucosylated Rac1 suggests that the autoprocessing mutants are still able to modify Rac1 in cells.