EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

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.

Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication

“…EGA did not completely inhibit intoxication with DT but delayed it, which was expected from our earlier results with EGA and binary clostridial toxins [ 35 ] and with DT and other pharmacological inhibitors of toxin uptake [ 5 ]. The reason is most likely related to the extreme potency of AB-toxins in cells, as few molecules of their catalytic moieties in the host cell cytosol is usually sufficient to exhibit the full cytotoxic effects [ 37 ].…”

“…Moreover, our findings suggest that EGA inhibits the pH-dependent transport of DTA across membranes into the cytosol. This step was also inhibited by EGA in the case of clostridial binary toxins, which also deliver their A subunit from acidic endosomes to the cytosol [ 35 ]. All toxins which were inhibited by EGA, exploit acidic endosomal vesicles for cellular trafficking, suggesting a common inhibitory mode of action for EGA in cells.…”

“…The compound 4-bromobenzaldehyde N -(2,6-dimethylphenyl)semicarbazone (EGA) was previously identified as a potent inhibitor that protects cells from toxins and viruses which enter the cytosol of cells via acidified vesicles [ 33 , 34 ], such as the Bacillus anthracis lethal toxin and DT [ 34 ], as well as the binary actin ADP-ribosylating toxins C2 from Clostridium ( C. ) botulinum , iota from C. perfringens and CDT from C. difficile [ 35 ]. EGA also protects neuronal cells from C. botulinum neurotoxins [ 36 ] and it was suggested that this compound might modulate intracellular toxin trafficking [ 34 , 35 , 36 ]. Prompted by these findings, we analyzed the effect of EGA on the intoxication of HeLa cells with DT in more detail.…”

Regulation of endo‐lysosomal pathway and autophagic flux by broad‐spectrum antipathogen inhibitor ABMA

“…Small-molecule inhibitors of intracellular trafficking have emerged in cell biology studies to explore cellular physiology as well as pathogenesis of various disease-inducing agents, especially pathogenhost interactions [16,18,20,21,39,40]. Compared to dominant-negative or genetic modulation of specific proteins involved in trafficking, chemical reagents harbor particular advantages to regulate trafficking, e.g.…”

“…These molecules, without exception, exhibit broad-spectrum anti-pathogen activities [8,11,18,20]. Furthermore, these specific molecular tools contribute substantially to the understanding of intracellular trafficking and infection mechanisms of known as well as emerging pathogens [11,18,21].…”