Botulinum toxin

Introduction:

Botulinnum toxin is a protein and is the most potent toxin known; it is produced by rod shaped bacteria, Clostridium botulinum. It's toxic effects can result in flaccid paralysis along with side effects such as vomitting, nausea and dizziness and can result in death. The bacteria can be found in soil, aquatic sedimants, the gastrointestinal tract of birds, fish and animals and may also be found in or on raw food. There are seven types of the botulinum toxin (A - G) with A, B, E and F being the most prevalent in humans. Three categories of botulism can occur in humans- foodborne botulism, wound botulism and infant botulism. Although toxic, it may also be used as a therapeutic tool in the treatment of neurological and opthalmic disorders caused by unusual and unnecessary muscle contractions as well as cosmetically.

Structure:
Botulinum toxin is a protein produced by the bacteria, Clostridium botulinum. It exists in bacteria as a single-chain protein until it undergoes post synaptic cleavage by protease endogenous or exogenous enzyme. This cleavage occurs between two cysteine residues (a disulphide bridge). This results in neurotoxin activation and the protein is divided into two separate chains- a light chain and a heavy chain. The light chain is half the size of the heavy chain and contains a zinc binding motif. This motif consists of two histidine residues, a glutamate residue and a water molecule. The zinc binding motif is a zinc dependent endopeptidase that works by cleaving peptide bonds of amino acids that are found inside a molecule. The heavy chain consists of a C–terminal that attaches to the surface of target cells and an N-terminal that is involved in the translocation of the light chains across membranes.

Image 1: Structure of botulinum toxin B
Image 1: Structure of botulinum toxin B

Mechanism of Action:

In all cases of the botulinum toxins, the toxins inhibit the release of acetylcholine. The mechanism of action of botulinum toxin involves four steps:

i)Binding.
In order for binding to occur the neurotoxin must move from the site of production to the presynaptic membrane terminals by diffusion. The toxin binds to the presynaptic membrane with high affinity. This binding occurs through the C-terminal in the heavy chain which is selective for the cholinergic nerve terminal. The toxin binds to a double-receptor model. It binds to the first receptor by lock and key and this brings it in contact with the second receptor. It is believed that the second receptor is an integral membrane protein and that possible molecules for receptor two include synaptotagmin 1 and 2. This binding will only occur if polysialogangliosides are present.

ii)Internalization.
The toxin is unable to enter the cell directly through the plasma membrane. Instead it must enter by endocytosis through acidic cellular compartments. The toxin enters into the lumen of the vesicle by a temperature and energy dependent process. Once internalized in the vesicle, the heavy chain forms a channel which releases the light chain is released into the cytoplasm.

iii)Membrane translocation.
This step involves the movement of the light chain from endosome to the cytosol. It in the cytosol that the light chain with perform its proteolytic activity. However in order for this step to happen the light chain must be exposed to a low pH which will result in a conformational change. This conformational change causes in the structure becoming acidic as hydrophobic areas become exposed on the surface. Once inside the toxin form channels that open at a pH of 5 and are non-selective. These channels are involved in the translocation of the light chain. When at low pH the light chain unfolds and is transported through the pores of the heavy chain. Once it has passed through transmembrane the light chain refolds and is released from the vesicle due to reduction of the disulphide bonds. Following this the heavy chain becomes tightly bound again and exits the membrane.

iv)Enzymatic target cleavage.
VAMP, SNAP-25 and syntaxin are SNARE proteins that are targeted by the toxin. The zinc dependent endopeptidase cleaves their peptide bonds and this results in proteolysis. This degradation prevents vesicle fusion by exocytosis which is promoted by the SNARE proteins. These proteins are only able to be cleaved by the zinc dependent endopeptidase before the SNARE proteins are formed.












Flaccid Paralysis:
Flaccid paralysis of voluntary muscles is the main symptom of botulism. It occurs due to the blocking of acetylcholine at peripheral cholinergic nerve endings. This causes the contraction of muscles under the skin. The muscles are paralyzed and this prevents the movement of skin. In cosmetics, when botox is administered to patients the muscles the muscles start to relax after paralysis which causes the disappearance of wrinkle on the skin; denervation occurs and causes the paralysis which gives the smooth appearance to the skin.

Image 2: Flaccid paralysis and ptosis.
Image 2: Flaccid paralysis and ptosis.

Symptoms:
The symptoms of botulism can sometimes result in a misdiagnosis due to their similarity with other illnesses, e.g. food poisoning caused by Salmonella. The symptoms of botulism tend to appear more rapidly with toxins B and E. The main symptom of botulism is flaccid paralysis without the presence of sensory abnormalities and autonomic dysfunction.

In foodborne botulism, the symptoms generally don’t appear until twelve to thirty-six hours later. The preliminary symptoms include nausea, tongue weakness, a reduction in the gag reflex and a weakness to cranial muscle which is followed by a weakness to the torso, muscles of respiration and the lungs. The preliminary symptoms are then followed by a respiratory depression and symmetrical limb weakness.

In the case of infant botulism the main preliminary symptoms are constipation and a difficulty in feeding. In severe cases the infant can have excessive drooling, a loss of head control and flaccid paralysis.

The symptoms of wound botulism are very similar to foodborne botulism such as disphagia and dysphoria. However premonitory symptoms are lacking in wound botulism. Wound botulism doesn’t result in outbreaks like foodborne botulism as it not food the is contaminated and only the penetrating wound found on the patient. Wound botulism is rare and this often causes it to be misdiagnosed with gangrene or tetanus even though neither shows signs of flaccid paralysis.


Image 3: Infant botulism. The infant cannot hold his head upright and is unable to suck or swallow.
Image 3: Infant botulism. The infant cannot hold his head upright and is unable to suck or swallow.

Treatment:

If diagnosed early, botulism can be treated with trivalent equine botulism antitoxin that is effective against the neurotoxins A, B and E. The antitoxin is administered intravenously and intramuscularly. Antitoxin is not usually administered for the treatment of infant botulism. Instead BabyBIG (botulism immune globulin intravenous) has been developed which can be administered intravenously to infants. BabyBIG is very expensive and as a result studies have found a new alternative treatment to infant botulism, equine botulism antitoxin (EqBA). It is safe and effective and should be used as an alternative treatment when BabyBIG is unavailable.

Patients diagnosed with food-borne botulism may also try removing the toxin from the gastrointestinal tract by inducing vomiting or else by the use of enemas. Neuromuscular Blockade Antagonists that cause the release of acetylcholine such as guanidine and aminopyridines have been investigated as potential treatment of botulism. The outcomes to date have be mixed, however studies have found that certain drugs may have an immunotype-specific ability to setback or avoid paralysis if given soon enough.

Therpaeutic Uses:
The botulinum toxin can be used therapeutically by injecting it into muscles in order to treat problems such as incontinence, abnormal head position, neck pain and blepharospasm. However the toxin is most commonly used as a cosmetic to remove facial wrinkles. The drug used is botulinum toxin A. It is injected into the muscles and this results in paralysis of the skin. The paralysis can last from three to four months. It is most comonly used to cause paralysis to areas of skin around the eyes and forehead. The injections are administered in small doses in order to cause a weakness to the skin but without completely paralysing the skin.
Image 4: The effects of the botulinum toxin as a cosmetic on the skin
Image 4: The effects of the botulinum toxin as a cosmetic on the skin
Try our MCQ to see what you learned about the botulinum toxin!
Questions:
https://docs.google.com/spreadsheet/viewform?formkey=dDJaR3prZFZlb2VvNEl4Z3FoNkJ0QkE6MQ
Answers:
https://docs.google.com/spreadsheet/ccc?key=0AnjOj8t9XY4zdDJaR3prZFZlb2VvNEl4Z3FoNkJ0QkE

References:

Simpson, L.L., (1989) Botulinum Neurotoxin and Tetanus Toxin, California: Academic Press.
Bell, C., Kyriskides, (2000) Clostridium botulinum,United Kingdom: Blackwell Science.
(2011), Pubmed Health, http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001624/ [Accessed at: 17/02/2011. 16.31p.m.]

Structure:
(2007), Neuromuscular disease center,http://neuromuscular.wustl.edu/nother/bot.htm [Accessed at: 19/02/2012, 12.02p.m.]
Image 1: Patocka, J., Splino, M., (2002), Botulinum toxin: From poison to medicinal agent, http://www.asanltr.com/newsletter/02-1/articles/Botulinum.htm [Accessed at: 23/02/2012, 13.12p.m.]

Mechanism of Action:
Pellizzari, R., Ornella, R., Giampietro, S., Montecucco, (1999) Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses,The Royal Society.

Flaccid Paralysis:
Turkington, C., Dover, M.D., The Encycolpedia of skin and skin disorders, 3rd edition: Blackwell.
Image 2: Simpson, L.L., (1989) Botulinum Neurotoxin and Tetanus Toxin, California: Academic Press.

Symptoms:
Davis, C.P.,http://www.medicinenet.com/botulism/page4.htm [Accessed at: 22/02/2012, 14.35p.m.]
Image 2: http://www.infantbotulism.org/images/floppybaby.jpg [Accessed at: 25/02/2012, 21.40p.m.]

Treatment:
Vanella de Cuetos, EE., Fernadez, R.A., Bianco, M.I., Sartori, O.J., Piovano,M.L., Luquez, C., de Jong, L.I., (2011)http://www.ncbi.nlm.nih.gov/pubmed/21918119 [Accessed at: 22/02/2012, 17.30p.m.

Therapeutic Uses:
(2012) [[http://emedicine.medscape.com/article/1271380-overview#aw2aab6b4[Accessed|http://emedicine.medscape.com/article/1271380-overview#aw2aab6b4[Accessed]] at: 26/20/2012, 20.20p.m.]
Image 4:
http://www.lidlift.com/images/examples/fillers/fillers3/Restylane-example-3.jpg [Accessed at: 26/02/2012, 20.46p.m.]