title: Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca²⁺ and GTPγS.
abstract: Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTPγS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (Cm) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell "stimulated" Cm increase, as well as on "constitutive" secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3-24h exhibited a significant reduction of the mean absolute and relative Cm increase in response to free-Ca(2+) and GTPγS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity.
authors.affiliation: Department of Toxicology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany; Department of Neurology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: Balletta.Andrea@mh-hannover.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Balletta
authors.affiliation: Department of Cellular Imaging and Electron Microscopy, Leibniz Institute of Molecular Pharmacology, Robert Rössle Str. 10, 13125 Berlin, Germany. Electronic address: lorenz@fmp-berlin.de.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Lorenz
authors.affiliation: Department of Toxicology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: Rummel.Andreas@mh-hannover.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Rummel
authors.affiliation: Department of Toxicology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: Gerhard.Ralf@mh-hannover.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
authors.affiliation: Department of Toxicology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: Bigalke.Hans@mh-hannover.de.
authors.forename: Hans
authors.initials: H
authors.lastname: Bigalke
authors.affiliation: Department of Neurology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: Wegner.Florian@mh-hannover.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Wegner
mesh_terms: D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003956:Dialysis; D025802:Electric Capacitance; D005089:Exocytosis / Q000187:drug effects*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006596:Hexosaminidases / Q000378:metabolism*; D006801:Humans; D019410:Interleukin-16 / Q000378:metabolism*; D008407:Mast Cells / Q000187:drug effects* / Q000378:metabolism; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D019410:Interleukin-16; C031815:toxB protein, Clostridium difficile; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006596:Hexosaminidases; D002118:Calcium
keywords: Clostridium difficile toxin B (TcdB); GTPγS; Hexosaminidase; Human mast cell line-1 (HMC-1); Membrane capacitance; Whole-cell patch-clamp