CLOSTRIDIUM-DIFFICILE TOXIN-B ACTIVATES CALCIUM INFLUX REQUIRED FOR ACTIN DISASSEMBLY DURING CYTOTOXICITY

The principal cellular response to Clostridium difficile toxin B, a pr otein toxin associated with antibiotic-associated colitis, is the disa ssembly of actin microfilaments. Although receptor-activated signal tr ansduction mechanisms have been proposed to mediate these effects, the intracellular events that precede actin breakdown are unknown. In NIH -3T3 fibroblasts, toxin B induced an elevation of intracellular calciu m possessing either a slow (minutes) or fast (seconds) rise time, foll owed by a sustained elevation of calcium concentration. Subcellular an alysis of steady-state calcium distribution after toxin B demonstrated that the increase of calcium was homogeneous throughout the cytosol a nd did not vary based on the kinetics of the initial calcium rise. Ah calcium responses were blocked by substitution with calcium-free buffe r or buffer containing lanthanum chloride, indicating that the rise in calcium was attributable to calcium influx from the extracellular spa ce. Quantitatively similar responses were observed in primary cultured gastric smooth muscle and AR42J pancreatic tumor cells, suggesting th at toxin-induced calcium signal transduction was conserved between cel l types. The morphological response to toxin B consisted of sequential dissociation of the actin cytoskeleton from membrane attachments, ret raction of actin stress fibers from the periphery to the perinuclear r egion, loss of fiber alignment, and cell rounding. The actin reorganiz ation associated with term B was blocked by incubation of cells in cal cium-free media or the clamping of intracellular calcium with cell-per meant calcium chelating agents. These results demonstrate that the cal cium influx activated by C. difficile toxin B is a necessary condition for the breakdown of filamentous actin associated with cytotoxicity.