Abstract
The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.
Highlights
The pathogenic clostridia Clostridium (C.) difficile, C. perfringens and C. botulinum produce the binary protein toxins CDT [1,2,3,4], iota [5,6,7,8] and C2 [9,10,11], respectively, which enter mammalian cells and directly modify the actin cytoskeleton, which results in cell-rounding and, apoptotic cell death
Vero cells were treated with each of the binary toxins in the presence or absence of EGA, and thethe intoxication process was monitored whichisisaawell‐established, well-established,highly highly intoxication process was monitoredininterms termsof ofcell-rounding, cell‐rounding, which specific and sensitive endpoint to detect the uptake of the components of these toxins into the host specific and sensitive endpoint to detect the uptake of the A components of these toxins into the host cellcell cytosol
We have found that the compound EGA delays the intoxication of different cultured mammalian cells with the binary clostridial toxins CDT, iota and C2, but not with the C. difficile toxins A and B
Summary
The pathogenic clostridia Clostridium (C.) difficile, C. perfringens and C. botulinum produce the binary protein toxins CDT [1,2,3,4], iota [5,6,7,8] and C2 [9,10,11], respectively, which enter mammalian cells and directly modify the actin cytoskeleton, which results in cell-rounding and, apoptotic cell death. These toxins are composed of two separate proteins, which must form complexes on mammalian. ADP-ribosylating toxins CDT, iota and C2, which require trafficking through acidified endosomal vesicles in order to identify a novel pharmacological inhibitor against this toxin family
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