Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca²⁺ and GTPγS.

Andrea Balletta, Dorothea Lorenz, Andreas Rummel, Ralf Gerhard, Hans Bigalke, Florian Wegner

Journal: Toxicology 2015;328():48-56

PMID: 25497110

Full Text on Elsevier Science View on PubMed

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.

Address: 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: [email protected].; Department of Cellular Imaging and Electron Microscopy, Leibniz Institute of Molecular Pharmacology, Robert Rössle Str. 10, 13125 Berlin, Germany. Electronic address: [email protected].; Department of Toxicology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: [email protected].; Department of Toxicology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: [email protected].; Department of Toxicology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: [email protected].; Department of Neurology, Hannover Medical School, Carl Neuberg Str. 1, 30625 Hannover, Germany. Electronic address: [email protected].

DOI: 10.1016/j.tox.2014.12.006

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MeSH Terms: Bacterial Proteins, Bacterial Toxins, Calcium, Cell Line, Dialysis, Electric Capacitance, Exocytosis, Guanosine 5'-O-(3-Thiotriphosphate), Hexosaminidases, Humans, Interleukin-16, Mast Cells, Membrane Potentials, Patch-Clamp Techniques, Time Factors