Botulinum Neurotoxins


Botulinum neurotoxins (BoNTs) are Janus‐faced biological agents. They are the most poisonous substances known and the causative agents of botulism, a deadly neuroparalytic syndrome of animals and humans. Owing to their potency, BoNTs have the potential to be used as biological weapons. At the same time, they are effective therapeutics for a variety of human neurological disorders and aesthetic medicine. In addition, the understanding of BoNT mechanism of action has provided great contributions to the study of the general principles of neuronal physiology. BoNTs were discovered more than a century ago, but they are still actively studied, both to identify effective countermeasures and mostly to expand the landscape of their clinical use. BoNT nomenclature has been recently revised owing to the identification of several BoNT isoforms via next‐generation sequencing techniques. The reason behind such variability among BoNTs remains elusive.

Key Concepts

  • Botulinum neurotoxins are exotoxins produced by neurotoxigenic bacterial strains of the genus Clostridium.
  • Botulinum neurotoxins are metalloprotease causing botulism, a deadly neuroparalytic syndrome affecting vertebrates.
  • Botulinum neurotoxins are a large and growing family of variants called serotypes, subtypes or mosaics according to their immunological and amino acid composition.
  • In spite of their variability, botulinum neurotoxins have a highly conserved structure and a common mechanism of action.
  • Botulinum neurotoxins block neurotransmitter release at peripheral nerve terminals causing flaccid paralysis and autonomic dysfunctions.
  • Botulinum neurotoxins are Janus‐faced biological agents, being potential bioweapons and very effective therapeutics at the same time.
  • Botulinum neurotoxins type A1 and B1 are used in human therapy to attenuate hyperactive peripheral nerve terminals.
  • Botulinum neurotoxin type A1 is a best seller treatment in aesthetic medicine.

Keywords: Clostridium; botulinum neurotoxin; botulism; neuromuscular junction; neuroexocytosis; bioterrorism; peripheral nervous system

Figure 1. Scheme of Botulinum Neurotoxin serotypes, subtypes and BoNT‐like toxins. Serotype classification was done on the basis of immunological methods, whereas subtypes have been mostly identified via gene sequencing methods. The tree was generated using ClustalW.
Figure 2. Classification of human botulism. There are five forms according to the mode of BoNT entry into the body. Food‐borne botulism occurs following the ingestion of BoNT‐containing foods. Infant botulism is caused by the ingestion of spores that germinate into the gastrointestinal tract owing to a lack of competition from the resident microbiota. These are the most common forms. The other three are much rarer and include inhalational botulism after inhalation of BoNT‐containing aerosols, iatrogenic botulism that occurs after the improper use of therapeutic BoNT, and wound botulism almost exclusively associated with drug injection. Any of these forms leads to toxin entry into circulation and eventually intoxication of peripheral nerves causing the flaccid paralysis of botulism.
Figure 3. Scheme of botulinum neurotoxin gene clusters and of progenitor toxin complex. Panel a displays the toxin gene arrangements of BoNT/A1 from C. botulinum strain Hall and BoNT/B1 from C. botulinum strain Okra, the two BoNTs used in human therapy. These gene clusters are characterised by the presence of the ha operon encoding for the HA proteins. The toxin gene cluster from C. botulinum strain Kyoto producing BoNT/A2 is reported to show the arrangement of the orfX operon. The ntnha gene is invariably present, along with genes expressing regulatory proteins like bot/R. The function of the p47 gene product is not known. Panel b shows a cartoon of the protein products of the bont/a1 gene cluster. BoNT and NTNHA form a heterodimer that assembles in a PTC together with the tripod formed by the combination of the three HA proteins.
Figure 4. Intestinal absorption of the BoNT and BoNT complexes. PTC (a), BoNT‐NTNHA complexes (b), or BoNT alone (c) can be adsorbed by transcytosis upon interaction with glycans and/or unknown receptors expressed on the intestinal wall. After transcytosis, PTCs are released in the basolateral surface, where they are expected to disassemble (a1). This frees the BoNT, which can enter the general circulation and the HA complex that is believed to disrupt cell–cell adhesions (a2) by sequestration of E‐cadherins (red rods). This could open a paracellular route for BoNTs and PTCs entry (black arrow). Alternatively, HA proteins may interact with cell–cell junction directly from the apical side of the intestinal barrier (d). After the passage across the intestinal wall, BoNTs enter the general circulation wherefrom they reach peripheral nerves.
Figure 5. BoNT structure and mechanism of action at nerve terminals. (a) Organisation of the mature BoNT molecule in two chains showing the three main domains with specific functions. The cysteine residues forming the interchain disulfide are depicted by two orange circles. The L chain contains the HEXXH motif of metalloproteases. (b) A space‐filling model is showing the structural architecture of the BoNT molecule. The L chain (red) is linked to the HN domain (yellow) via the interchain disulfide bond (inset shown in cartoon mode with the two sulfur atom represented by orange spheres) and a peptide belt (shown in dark blue) that surrounds the L domain. The HC is divided into two subdomains, the HC‐N (violet) and the HC‐C (green). (c) Nerve terminal intoxication by BoNTs is divided into 5 main steps. (1) HC domain (green) binds to a polysialoganglioside (PSG) receptor in anionic microdomains present on the presynaptic membrane. This facilitates the interaction with the secondary receptor, either synaptotagmin (pink rod) or glycosylated SV2 (black). (2) The BoNT is then internalised inside SVs and trafficked within the nerve terminal. (3) The v‐ATPase (orange) induces the acidification of the vesicle (redshift of the lumen) to drive the refilling of neurotransmitters (blue dots). Acidification triggers the membrane translocation of the L chain into the cytosol, which is assisted by the HN domain (yellow). (4) The L chain (red) is released from the HN domain by the action of the thioredoxin reductase–thioredoxin system (TrxR‐Trx, blue and dark blue) and Hsp90 (violet), which reduce the interchain disulfide bond (orange) and avoid the aggregation of the protease. (5) The L chain is released in the cytosol where it displays its metalloprotease activity: BoNT/B, /D, /F, /G /X cleave VAMP (blue); BoNT/A and BoNT/E cleave SNAP‐25 (green); and BoNT/C cleaves both SNAP‐25 and syntaxin (red).


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Further Reading

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Masuyer G, Chaddock JA, Foster KA and Acharya KR (2014) Engineered botulinum neurotoxins as new therapeutics. Annual Review of Pharmacology and Toxicology 54: 27–51.

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Sudhof TC and Rizo J (2011) Synaptic vesicle exocytosis. Cold Spring Harbor Perspectives in Biology 3 (12).

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Marco, Pirazzini, and Rossetto, Ornella(Sep 2020) Botulinum Neurotoxins. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0029189]