Remember my past post on botulinum toxin, the deadliest substance known to mankind? Well, it’s time to visit the second runner up. Tetanospasmin is a neurotoxin created by another member of the Clostridium genus: Clostridium tetani. That is one genus you don’t want to mess with! This particular member of Clostridium is the cause behind all those lovely tetanus shots and boosters you have been subjected to through the years. When you consider the fact that you’re being vaccinated against the second deadliest toxin in the world, maybe you won’t mind the needle jab quite as much. In a rather bizarre twist, the neurotoxin has no apparent use or benefit to C. tetani in its natural environment. The intoxication of other organisms, such as us, appears to be mainly incidental.
Meet the Maker: Clostridium tetani
Thanks to producing the top two deadliest toxins, the Clostridium genus is one of the most infamous groups of anaerobic pathogens out there, causing botulism, tetanus, gas gangrene, and necrotic enteritis . The species that causes tetanus is Clostridium tetani, a Gram positive, obligate anaerobic, rod-shaped bacterium. It can be found worldwide in dirt and feces and also winds up in house dust, contaminated heroin, and even our gastrointestinal tract [1,7].
During its growth phase, the bacterium is motile and has multiple flagella (lash-like appendages used for movement). At this age, it produces the toxins tetanospasmin and tetanolysin, which we will explore in the next section. When it fully matures, the flagella drop off and it develops a spore at one end, causing it to look like, to quote the literature, “a drumstick.” These spores can survive oxygen exposure, unlike the other phases of the microbe, and are also resistant to environmental extremes and chemical agents such as formalin and ethanol. They remain in this protective spore phase until conditions, such as very low oxygen exposure, promote germination. These spores are what can be found in feces and soil, and can be become caught in puncture wounds, scratches, burns, and other injuries. Once the conditions become anaerobic (if they’re caught in a wound this occurs when the wound closes or secreted fluids encase the spore), the spores mature into vegetative forms that multiply and release tetanospasmin and tetanolysin once again [1,2].
Tetanospasmin Neurotoxin: The Toxin
Tetanospasmin is one of the most powerful neurotoxins known, with the lethal dose being a mere 175 nanograms for a 154 lb (70 kg) human. As with its cousin botulinum toxin, this lethality is due to its incredibly high specificity and irreversible effects (recovery can only occur through new growth of the nerve cell). The gene that encodes this toxin is found on a large plasmid within C. tetani’s genome [3,4].
The tetanospasmin toxin is a zinc-dependent metalloproteinase (an enzyme that requires the metal zinc to function) whose molecular structure contains both a heavy and a light chain of molecules joined together. When the toxin enters the body, the heavy chain section forms a transmembrane pore in a nerve cell, allowing the light chain to enter the cell and cause toxic effects. The heavy chain section of the molecule is non-toxic, containing a section called Fragment C, and is used to facilitate the transport of the toxin from the peripheral nerves to the central nervous system. Inside of the intoxicated nerve, the light chain section targets the SNARE complex, cutting synaptobrevin, a protein essential to the complex (In contrast, botulinum toxin cleaves syntaxin and SNAP-25, two proteins also involved in the SNARE complex). By cutting this protein, the SNARE complex can no longer function: vesicles containing neurotransmitters will not be released from the nerve, preventing the initiation and inhibition of muscle contraction [1,3,4,6]. This pathway was explained in The Toxin section of this post.
Because the first nerves the toxin will encounter after entering through a wound will be motor neurons causing muscle contraction, the first toxic effect is a flaccid paralysis identical to that of botulism and botulinum toxin. However, tetanospasmin differs from botulinum toxin in that it travels up the axon of nerves toward the central nervous system, infecting cell bodies of neurons in the brainstem and spinal cord. It has a higher affinity for, and therefore preferentially targets, inhibitory neurons that release the neurotransmitters GABA and glycine. These neurotransmitters prevent muscle contraction. This inhibition of contraction is important during movement because, for example, when you flex your bicep, the antagonistic muscle (your triceps) is forced to relax in order to allow the bicep contraction to occur. Inhibitory neurons prevent the antagonist muscles from contracting when their partner muscle contracts. If an antagonistic muscle co-contracts, it becomes extremely difficult to move because the two muscles are contracting to pull your body in opposite directions! Consequently, the characteristic effect of tetanospasmin is to cause a rigid paralysis: sustained muscle contractions along with painful and uncontrollable muscle spasms. Once the toxin has invaded a nerve and cut the SNARE complex, the effects are permanent. The body will eventually grow new nerve terminals to replace the inactive ones, but the process can take at least six to eight weeks [1,3,4,5,6].
C. tetani also produces the toxin tetanolysin, which damages tissue cells and aids in creating an anaerobic environment for the spores to germinate in . It isn’t shockingly deadly enough to warrant any more attention in this post, however.
Tetanus: The Intoxication
Once intoxicated with tetanospasmin, humans suffer from the condition known as tetanus. This is a nervous system disorder that is one of the most common, fatal infectious diseases in the world. While it is completely preventable with vaccinations, it still has a high worldwide mortality rate. In the United States, where most of us are vaccinated, we get about 31 cases a year (a nice drop from the 1940’s, when there were 500-600 cases a year), with a case fatality of 13% . However, the global incidence is about 1 million cases a year with a 30-50% mortality rate. In countries where medical treatment is not adequate, the mortality rate hovers around 100% . Between 800,000 and 1 million deaths are caused by tetanus each year, and about half of those are attributed to neonatal (infant) cases. Most of these fatal cases occur in Africa and South East Asia due to a lack of supportive care; the rigid paralysis causes breathing problems, and ventilation support reduces the mortality risk from 65-90% to 10% in neonatal cases . On top of vaccinations, approximately 30% of the population has a natural immunity or disease resistance to tetanus !
Once tetanospasmin has entered the human body through an injury, there is an incubation period during which the spores await conditions to transform into bacteria and then bacterial toxin secretions build up. There is no visible sign of infection in the wound, and many times the injury is healed by the time symptoms start to present. The incubation period averages about 8 days, with the length directly proportional to the distance between the wound and the central nervous system (a head or neck injury will present faster than if the spores entered a cut on a finger) [1,2,5].
A person suffering from tetanus will experience spasms and increasing muscle rigidity for the first week, followed by autonomic disturbances (breathing, heartbeat, salivation, digestion, etc) which peak in the second week and can last for one to two weeks. Muscle spasms often subside after three weeks, while the muscle rigidity may continue for six to eight weeks. The classic symptoms are trismus (lockjaw), caused by muscle spasms in the Masseter muscle of the jaw, and risus sardonicus (literally, ‘sardonic smile’) due to facial muscle spasms. The muscle spasms can be violent enough to cause bone fractures (including spinal fractures) and tendon avulsions. Additionally, sensory nerves can be invaded by the toxin and cause altered sensations, such as pain [1.2.5]. It is not known whether or not the toxin spreads from the brainstem into areas of the brain responsible for higher cognitive function. Generally, adult survivors show no sign of cognitive symptoms; however neonatal tetanus can result in intellectual disability . In most cases, if patients survive, they reach a complete recovery; occasionally there are long-term effects, including limb contractures, bed sores, seizures, and sleep disturbances.
There are four clinical forms of tetanus, classified based on the extent and location of the neurons that become intoxicated  . The most common form is generalized tetanus, in which patients suffer the characteristic lockjaw and risus sardonicus as well as further tonic contractions of the skeletal muscles and intense muscle spasms. Laryngeal and diaphragm paralysis can cause apnea and breathing problems requiring ventilation support and pain, difficulty swallowing, and stiffness are the most common complaints. As tetanus progresses, more muscle groups become paralyzed, with contractions causing vertebral and bone fractures and hemorrhaging in the muscles. These spasms can be triggered by anything from changes in light, voices, and air drafts. The major cause of death in this form is due to increased autonomic over activity and instability: the toxin causes problems with heartbeat, breathing, fever, and cardiac arrest [1,2] .
The other three forms of tetanus are less common. Local tetanus occurs when antitoxins are administered and prevent the spread of the toxin throughout the body, limiting the damage to the local site of injury. Patients of local tetanus will suffer steady and painful muscle contractions and spasms at the site of the injury, which may linger for several weeks to months before completely healing. In these cases, the fatality rate is only 1% as the autonomic functions are rarely impacted. Cephalic tetanus, the third type, is a variation of local tetanus that usually occurs if the infected wound was on the head. The resulting paralysis is limited to the cranial muscles. Finally, neonatal tetanus occurs when infants become infected, often through umbilical stump infections (some countries traditionally apply poultices such as cow dung and ashes to the umbilical cord which skyrockets the risk of tetanus) or due to the mother not being immunized. This is more common in developing countries, and has a staggering 50-100% mortality rate due to the high load of toxin causing death by breathing problems or sepsis [1,2].
Diagnosis, Treatment, Prevention
The diagnosis of tetanus is clinical, thanks to the highly characteristic features of the disease. Laboratory studies are used to eliminate similar conditions, but there is no definitive laboratory test for tetanus because C. tetani is only recovered in about 30% of cases, with wound cultures often turning up negative. Additionally, patients can be found to carry C. tetani, but they might be immune to the toxin and not actually have tetanus .
Once diagnosed, the treatments focus on preventing further toxin release, neutralizing any free toxin molecules, and minimizing effects of cleaved SNARE complexes. To prevent further toxin release, antibiotics are used to kill C. tetani. While penicillin is a common antibiotic, it is a GABA antagonist (it binds to and blocks GABA receptors, while the toxin blocks the release of GABA) and can therefore compound the muscle spasms and should be avoided. To neutralize unbound toxin, tetanus immunoglobulin is injected. There is no way to deactivate a bound toxin and reverse the damage done to SNARE complexes, so, instead doctors aim to control the seizures using sedation or reducing possible sources of provocation of muscles spasms (placing patients in dark and quiet rooms). Further, supportive care in the form of ventilation is often vital [1,2].
Optimally, preventative measures should be taken to immunize the population. The tetanus toxoid is used to provide an active immunization, recommended to be given every couple of years during childhood and supplemented by a tetanus booster every ten years in adults [1,5]. If you ever had qualms about vaccinations before, perhaps knowing they can protect you from the second deadliest substance known to man will make you appreciate them a little more next time!
Using a Toxin to Fight Intoxication
You may have noticed I mentioned botulinum toxin quite a bit throughout this post. It was not entirely shameless advertising of previous posts on my part; hopefully you picked up on the fact that these two related toxins cause opposite effects–botulinum toxin causes a flaccid paralysis while tetanospasmin causes a rigid paralysis. Additionally, botulinum toxin is far more confined to nerve terminals in motor neurons as it has no retrograde transport into the central nervous system. These differences are important because botulinum toxin could be used to counter the effect of tetanospasmin! In fact, in 6 cases of tetanus, doctors have used botulinum toxin A to control the painful muscle contractions. In 4 of the 6 cases, improvement was noted within 1 to 4 days of administration. Of course, the doctors have to be extremely careful to inject the botulinum toxin away from any vital structures such as arteries and the larynx .
This toxin-to-fight-toxin scenario would reduce the need for muscle relaxant drugs. Furthermore, as botulinum toxin is more long-lasting than tetanospasmin, one treatment should outlast the duration of the painful muscle spasms associated with the diseases. On the other hand, as nearly all skeletal muscles are affected in tetanus, it would be difficult to treat them all with botulinum toxin. Injecting the deadliest toxin into a human to help counter the second deadliest toxin’s effects has its drawbacks as well: risk of overdosing, possible side effects, and, of course, the cost . Still, the concept is pretty darned neat!
While botulinum toxin is notorious for being utilized as a biological weapon, tetanospasmin, despite being the second deadliest toxin, has managed to dodge that dubious honor. While the fatal dose is minuscule, the widespread vaccinations and natural immunity make the toxin a poor weapon choice. In fact, I can’t find any mention of it being aerosolized or tetanus ever being caused by inhalation. Rest easy, folks!
Toxin: Therapeutic Uses
Thanks to the unique structure of this toxin (specifically the C-fragment heavy chain allowing for retrograde transport up neurons and into the central nervous system) tetanospasmin is becoming a shining star in bio-therapeutics. As the C-fragment is not toxic, it has been adopted as a carrier to move therapeutic drugs to the central nervous system. Many neurological disorders such as Alzheimer’s, Parkinson’s, and Huntington’s all require very specific delivery of a drug. Normally this is a difficult problem due to the blood-brain-barrier, which prevents many drugs from entering the brain through the blood system. The use of virus machinery or the C-fragment of tetanospasmin allows the blood-brain-barrier to be bypassed, delivering drugs directly to the brain. Furthermore, research has shown that the C-fragment itself, without any drug attached, actually has therapeutic properties. In rat models of Parkinson’s, the binding of C-fragments to cells activates a neurotrophic factor pathway in the cell that protects against cell death, improves motor deficits, and restores dopamine levels to normal [4,9]!
Tetanospasmin is also being used in conjunction with a transcription factor from yeast to anatomically map the brain of fruit flies. Researchers are using the light chain of the toxin to specifically block synaptic transmission (communication between neurons in the brain) in order to discern the role of individual neurons in the brain and the impact they have on certain behaviors. This could not only provide us with a map of the circuitry in the brain, but help explain the neurological mechanisms behind certain behaviors !
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