Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells

Abstract

SummaryClostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdBtreated HeLa cells. Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects. A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity. The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB 1-556 ). Unlike TcdB treated cells, intoxication with TcdB 1-556 or expression of TcdB 1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB 1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspasedependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.

Clostridium difficile Toxin B Causes Apoptosis in Epithelial Cells by Thrilling Mitochondria

“…Apoptosis Induced by C. difficile Toxin B Is Mediated by Caspases 9 and 3-Although several lines of evidence have pointed to TcdB as a proapoptotic factor (11,12), the actual mechanism that leads to cell death is not yet precisely defined. Hence, to address this question, we first assayed which of the two main apoptotic pathways was involved.…”

“…difficile toxin B (TcdB) is another large clostridial toxin, largely accepted as a proapoptotic factor (11,12). It is produced, together with TcdA, by pathogenic strains of C. difficile, recognized as the principal cause of antibiotic-associated pseudomembranous colitis (reviewed in Ref.…”

Application of Mutated Clostridium difficile Toxin A for Determination of Glucosyltransferase-Dependent Effects

“…Two highly conserved motifs were chosen for mutation, namely, tryptophan-101 and the DXD motif at positions 285 to 287 (3,17). Analogous mutations of TcdB resulted in reductions of the in vitro GT activity, by factors of 1,000 and 5,000, respectively (5,6,16). The in vitro GT activities of rTcdA (wild type), rTcdA W101A (tryptophan mutant), and rTcdA D285/287N (DXD mutant) were determined from the linear phases of RhoA glucosylation kinetics (Fig.…”

Structural organization of the functional domains of Clostridium difficile toxins A and B

“…The proteins are homologous glucosyltransferases that inactivate small GTPases of the Rho/Rac family. The resulting disruption in signaling causes a loss of cell-cell junctions, dysregulation of the actin cytoskeleton, and apoptosis (4,5).…”