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A large number of bacterial toxins consist of active and cell binding protomers linked by an interchain disulfide bridge. The largest family of such disulfide‐bridged exotoxins is that of the clostridial neurotoxins that consist of two chains and comprise the tetanus neurotoxins causing tetanus and the botulinum neurotoxins causing botulism. Reduction of the interchain disulfide abolishes toxicity, and we discuss the experiments that revealed the role of this structural element in neuronal intoxication. The redox couple thioredoxin reductase–thioredoxin (TrxR‐Trx) was identified as the responsible for reduction of this disulfide occurring on the cytosolic surface of synaptic vesicles. We then discuss the very relevant finding that drugs that inhibit TrxR‐Trx also prevent botulism. On this basis, we propose that ebselen and PX‐12, two TrxR‐Trx specific drugs previously used in clinical trials in humans, satisfy all the requirements for clinical tests aiming at evaluating their capacity to effectively counteract human and animal botulism arising from intestinal toxaemias such as infant botulism.

Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localised. L translocation is accompanied by unfolding, and it has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase-Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of heat shock protein 90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that geldanamycin strongly synergises with PX-12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulfide reduction are strictly coupled. Indeed we found that the heat shock protein 90 and the Thioredoxin reductase-Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone-redox machinery which is exploited by clostridial neurotoxins to deliver their catalytic part into the cytosol.

Summary Tetanus and botulinum neurotoxins are di-chain proteins that cause paralysis by inhibiting neuroexocytosis. These neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, whose acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage was made accessible to investigation by making it to occur at the surface of neurons. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxin B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Based on these and previous findings, we propose a stepwise sequence of molecular events that lead from toxin binding to membrane insertion.

Summary Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localised. L translocation is accompanied by unfolding, and it has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase–Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of heat shock protein 90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that geldanamycin strongly synergises with PX-12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulfide reduction are strictly coupled. Indeed we found that the heat shock protein 90 and the Thioredoxin reductase–Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone-redox machinery which is exploited by clostridial neurotoxins to deliver their catalytic part into the cytosol.

Tetanus and botulinum neurotoxins are di-chain proteins that cause paralysis by inhibiting neuroexocytosis. These neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, whose acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage was made accessible to investigation by making it to occur at the surface of neurons. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxin B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Based on these and previous findings, we propose a stepwise sequence of molecular events that lead from toxin binding to membrane insertion.