title: Clostridium difficile toxin B activates calcium influx required for actin disassembly during cytotoxicity.
abstract: The principal cellular response to Clostridium difficile toxin B, a protein toxin associated with antibiotic-associated colitis, is the disassembly of actin microfilaments. Although receptor-activated signal transduction 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 calcium possessing either a slow (minutes) or fast (seconds) rise time, followed by a sustained elevation of calcium concentration. Subcellular analysis of steady-state calcium distribution after toxin B demonstrated that the increase of calcium was homogeneous throughout the cytosol and did not vary based on the kinetics of the initial calcium rise. All calcium responses were blocked by substitution with calcium-free buffer or buffer containing lanthanum chloride, indicating that the rise in calcium was attributable to calcium influx from the extracellular space. Quantitatively similar responses were observed in primary cultured gastric smooth muscle and AR42J pancreatic tumor cells, suggesting that toxin-induced calcium signal transduction was conserved between cell types. The morphological response to toxin B consisted of sequential dissociation of the actin cytoskeleton from membrane attachments, retraction of actin stress fibers from the periphery to the perinuclear region, loss of fibre alignment, and cell rounding. The actin reorganization associated with toxin B was blocked by incubation of cells in calcium-free media or the clamping of intracellular calcium with cell-permeant calcium chelating agents. These results demonstrate that the calcium influx activated by C. difficile toxin B is a necessary condition for the breakdown of filamentous actin associated with cytotoxicity.
authors.affiliation: Division of Gastroenterology, St. Elizabeth's Hospital.
authors.forename: R J
authors.initials: RJ
authors.lastname: Gilbert
authors.forename: C
authors.initials: C
authors.lastname: Pothoulakis
authors.forename: J T
authors.initials: JT
authors.lastname: LaMont
authors.forename: M
authors.initials: M
authors.lastname: Yakubovich
mesh_terms: D016475:3T3 Cells / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D002021:Buffers; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002240:Carbohydrate Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005753:Gastric Mucosa / Q000378:metabolism; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000648:ultrastructure; D013270:Stomach / Q000648:ultrastructure; D014407:Tumor Cells, Cultured
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002021:Buffers; C031815:toxB protein, Clostridium difficile; C028521:lanthanum chloride; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D007811:Lanthanum; D002118:Calcium