Abstract
Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.
Highlights
Clostridium difficile toxin A (TcdA) and toxin B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi a-toxin (TcnA) are important pathogenicity factors of the family of clostridial glucosylating toxins (CGTs)
Dynasore conferred resistance towards cell rounding in HeLa cells incubated with the prototypic member of the CGT family, C. difficile toxin B (Fig. 1B)
The importance of dynamin in the uptake of CGTs was tested with toxin B in the human colon adenocarcinoma cell line HT-29 (Cell Line Services, Eppelheim, Germany)
Summary
Clostridium difficile toxin A (TcdA) and toxin B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi a-toxin (TcnA) are important pathogenicity factors of the family of clostridial glucosylating toxins (CGTs). CGTs consist of at least four domains [5]. At the N-terminus, the glycosyltransferase domain is located [6], which modifies low molecular mass GTP-binding proteins of the Rho and/or Ras family by mono-O-glucosylation [7],[8] or mono-O-GlcNAcylation (atoxin) [9]. An adjacent cysteine protease domain releases the glucosyltransferase into the cytosol by autoproteolytic cleavage [10]. Target cell binding is mainly mediated by the C-terminal domain, which is characterized by repetitive oligopeptides (CROPs) [11],[12]
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