(A) Primary human foreskin fibroblasts (HFFs) pretreated with Hpa-SL-AOMK are protected from holotoxin-mediated cell rounding. HFFs treated with the catalytically inactivated C698A holotoxin or left untreated exhibit minimal cell rounding.
(B) Quantification of inhibitor effects on holotoxin-mediated HFF cell rounding. Pre-treatment with Hpa-SL-AOMK or Hpa-KL-AOMK was protective, while Cbz-EL-AOMK and the pan-cathepsin inhibitor JPM-OEt had no effect on toxin function. Data represents the mean of three experiments ± standard deviation.
(C) Addition of wildtype TcdB holotoxin to HFFs results in complete glucosylation of the Rho GTPase Rac1, as seen by Western blot with the glucosylation-sensitive α-Rac1 monoclonal antibody mAb102. Pre-treating the cells with Hpa-SL-AOMK or Hpa-KL-AOMK protected HFFs from toxin effector domain activity, while inhibiting cathepsin activity with JPM-OEt did not.
See also Figure S3.