Mechanism of Botulinum Toxin
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Mechanism of Botulinum Toxin

Botulinum neurotoxin (BoNT) is one of the most potent toxins that inhibit neurotransmitter release at the neuromuscular junction. The toxin is a microbial product synthesized by an anaerobic, gram-positive, spore-forming bacteria Clostridium botulinum whose natural habitat is soil. In addition to C. botulinum, unique strains of Clostridium baratii and Clostridium butyricum also have the capacity to produce BoNT. The basis of the phenomenal potency of BoNT is enzymatic. The toxin is a zinc-dependent protease that cleaves one or more of the fusion proteins by which neuronal vesicles release Ach (Acetylcholine) into the neuromuscular junction. This toxin acts preferentially on peripheral cholinergic nerve endings to block Ach release and is  both an agent that causes disease (i.e., botulism) as well as an agent that can be used to treat the disease (e.g., dystonia). The ability of BoNT to produce its effects is largely dependent on its ability to penetrate cellular and intracellular membranes. Thus, the toxin that is ingested or inhaled can bind to epithelial cells and be transported to the general circulation (Ref.1).

There are 3 main clinical forms of botulism: food borne, intestinal and wound related. Food borne botulism occurs when food contaminated by the preformed BoNT is eaten. Intestinal botulism occurs when spores are ingested and reproduce in the gastrointestinal tract that releases the toxin in situ (primary infection, secondary intoxication). Wound-related botulism occurs when anaerobic conditions within an abscess allow germination of C. botulinum spores. Most of these cases originate in wounds contaminated with soil or at injection sites in intravenous drug users. Regardless of how the toxin gets into the gut, it must reach the general circulation for its lethal effects to be manifest. Since the toxin is too large to pass through epithelial barriers by diffusion, it is actively transported through the gut lining. The process by which this occurs involves receptor-mediated transcytosis that does not damage the gastrointestinal tract (Ref.1). C. botulinum produces all seven known serotypes of the BoNT (A, B, CAlpha, CBeta, D, E, F and G), whereas C. baratii and C. butyricum produce only one serotype each (F and E, respectively). Each type is antigenically distinct with its own characteristics. BoNTs are secreted as a single polypeptide chain of about 150kDa each, which are cleaved endogenously or exogenously resulting into a 100 kDa heavy chain and a 50 kDa light chain linked through a disulfide bond (Ref.2). The botulinum toxin’s mode of action involves three steps: extracellular binding and internalization, membrane translocation and intracellular substrate cleavage, and blockage of Ach release. The heavy chain plays dual role in the toxic action of BoNTs, cell surface binding (binding domain), and translocation across membranes (translocation domain), while the light chain is responsible for the intracellular toxic activity. When the toxin reaches peripheral cholinergic nerve endings, there is again a sequence of membrane-penetrating events. Initially, the toxin binds to the surface of plasma membranes, and this is followed by receptor-mediated endocytosis and pH-induced translocation across the endosome membrane. The receptor for BoNT at the neuromuscular junction has not been unequivocally identified. However, a sialic acid-containing molecule, and possibly a ganglioside, is implicated in toxin binding, but there is no consensus on the exact role played by this molecule. Once inside the low-pH endosome, the light chain dissociates from the heavy chain and is released into the cytosol, where it acts as a zinc metalloprotease and cleaves SNARE (Soluble NSF-attachment protein receptor) proteins. Without functional SNARE complexes, the neurotransmitter Ach is not released into neuromuscular junctions, leading to paralysis of myosin filaments. Blockade of transmitter release accounts for flaccid paralysis and autonomic dysfunction that is characteristic of the disease botulism. Although the toxin acts preferentially on cholinergic nerve endings, it does have the ability to block exocytosis from other nerve endings as well. BoNT types-A and E act on SNAP25 (Synaptosomal-associated protein of 25 kDa); serotypes B, D, F, and G act on VAMP (Vesicle-Associated Membrane Protein), also known as synaptobrevin; and serotype C acts mainly on Syntaxin, although it can also cleave SNAP25 (Ref.1). By disrupting neurotransmission at cholinergic junctions in the autonomic nervous system, the toxin can also cause various forms of autonomic dysfunction. Its most life-threatening potential, however, is its ability to stop respiration by disrupting neurotransmission in diaphragm and intercostal muscles.
 
Patients with botulism typically present with difficulty seeing, speaking, and/or swallowing. Prominent neurologic findings in all forms of botulism include diplopia, blurred vision, often enlarged or sluggishly reactive pupils, dysarthria, dysphonia, and dysphagia. The mouth may appear dry and the pharynx injected because of peripheral parasympathetic cholinergic blockade. Sensory changes are not observed except for infrequent circumoral and peripheral paresthesias from hyperventilation as a patient becomes frightened by onset of paralysis. As paralysis extends beyond bulbar musculature, loss of head control, hypotonia, and generalized weakness become prominent. In untreated persons, death results from airway obstruction (pharyngeal and upper airway muscle paralysis) and inadequate tidal volume (diaphragmatic and accessory respiratory muscle paralysis). Because botulism is intoxication, patients remain afebrile unless they also have acquired a secondary infection (e.g., aspiration pneumonia). The toxin does not penetrate brain parenchyma; however, they often appear lethargic and have communication difficulties because of bulbar palsies (Ref.3). Complications, such as eyelid ptosis, have been attributed to BoNT-A (commonly referred to as Botox). An "hourglass" deformity, which is the consequence of temporalis muscle atrophy, has also been reported. In addition to acting as a toxin during botulism infection, Botox is also now being used to treat several disorders. Careful administration of very small doses of the toxin is used to treat two eye muscle disorders--uncontrollable blinking (blepharospasm) and misaligned eyes (strabismus) and a neurological movement disorder that causes severe neck and shoulder contractions, known as cervical dystonia (Ref.4). Botox not only affects the neuromuscular junction directly, but also have intrinsic pain controlling effects by acting on afferent pathways affecting pain perception. Recently Botox injection has come into vogue as a treatment for facial wrinkles. In the course of treating these wrinkles, it was discovered that people who suffered from migraines had a decrease in the frequency and severity of these types of headaches. Additional studies are necessary in order to validate the effects of BoNT in the treatment of migraines. Injection of botulinum toxin has also proven to be a safe and effective therapy for a variety of somatic and autonomic motor disorders. Urologists are now finding clinical success with urethral and bladder Botox injections in the treatment of detrusor-sphincter dyssynergia, non-neurogenic pelvic floor spasticity, and refractory overactive bladder (Ref.5). The main treatment for severe botulism is meticulous supportive therapy, which may include mechanical ventilation. To be most effective, the antitoxin must be given before much toxin has bound to presynaptic nerve endings. In cases of wound-related botulism, the wound must be debrided and therapy with an appropriate antibiotic such as penicillin started. Recovery takes weeks to months and occurs when new presynaptic end plates and neuromuscular junctions are formed. Although BoNT is used to reduce wrinkles and treat several disorders, BoNTs are also among the most lethal biological substances to have been weaponized as a highly toxic aerosol form. New vehicles for transmission have emerged in recent decades, and wound botulism associated with black tar heroin has increased dramatically since 1994. Such a potential bioterrorist threat necessitates the development of therapeutic countermeasures against BoNTs.