Clostridium botulinum C2 Toxin

Abstract

The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADPribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.

Clostridium botulinum C2 Toxin

“…This suggests that ion-ion interactions between the positively charged molecules and the channel-forming heptamers are at least partially responsible for their high affinity to the C2II channel (32). Channel block occurs also when C2I is added to the cis side of C2II reconstituted into lipid bilayers at very low C2I concentration (26). Binding of EF/LF to PA is also highly ionic strength-dependent, which is presumably caused by a number of negatively charged amino acids localized within the vestibule of the PA channel (35, 44 -46).…”

Binary Actin-ADP-Ribosylating Toxins and their Use as Molecular Trojan Horses for Drug Delivery into Eukaryotic Cells

“…It readily became evident that pore formation by C2IIa is absolutely dependent upon both acidic conditions and binding of C2IIa to the cellular receptor prior to acidification of the medium. C2I does not interact with membrane-inserted C2IIa pores [71], thus the amount of rubidium released from cells through the C2IIa pores does not decrease in the presence of C2I. Additionally, cells treated this way do not round up [71].…”

“…For example, pore formation by C2IIa in cell membranes can be measured independently of C2I by monitoring the efflux of radioactive rubidium-86 from preloaded cells [71]. It readily became evident that pore formation by C2IIa is absolutely dependent upon both acidic conditions and binding of C2IIa to the cellular receptor prior to acidification of the medium.…”

“…Then, during cellular uptake, acidic conditions within the endosomes induce a conformational change of C2IIa heptamers. Hydrophobic residues are exposed on the heptamer surface [71] and C2IIa inserts into endosomal membranes to form pores. Pore formation of C2IIa heptamers is absolutely essential for translocating C2I into the cytosol [71].…”

Inhibitors of pore-forming toxins

“…Similarly to the anthrax toxins, the cell-bound C2I/C2IIa, Ia/Ib, and CDTa/CDTb complexes are internalized by receptor-mediated endocytosis [62][63][64][65]. After endocytosis, these complexes reach endosomal vesicles where the A components translocate across the endosomal membranes into the cytosol, possibly using the C2IIa and Ib pores as translocation corridors [61,[65][66][67][68][69][70]. in vitro model bilayer experiments have showed that PA 63 is able to bind and translocate His-tagged C2I, while C2II binds, but does not translocate LF and EF [71].…”