Clostridium difficile toxin B induces reorganization of actin, vinculin, and talin in cultured cells (original) (raw)
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Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro
Infection and immunity, 1987
We describe a simplified procedure for purification of Clostridium difficile toxin B. In this procedure, cytotoxicity is associated with a single protein band with a molecular mass of 230 kilodaltons. We used direct fluorescent staining of actin filaments to study the effect of this toxin on cultured cells. Morphologic changes were preceded by a decrease in the number and length of stress fibers followed by their disappearance with condensation of cellular actin around the nucleus. We then showed that cells treated with either cytochalasin B or toxin B had a significant increase in the monomeric actin pool as quantitated by DNase I inhibition. In contrast to the cytochalasins, toxin B had no direct effect on the rate or extent of actin polymerization or network formation in vitro. Cytoplasmic extracts of toxin B-treated cells had a significantly lower level of modulating activity on actin assembly and interactions in vitro compared with extracts of untreated cells. These results sug...
Protein Expression and Purification, 2010
Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the
PLoS ONE, 2013
Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 -1852, designated as a ˜97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP 6 ), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-a) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.