April 24, 2019

By: List Labs

By: Hans Bigalke

Tetanus toxin (TeNT) and botulinum toxin (BoNT) appear quite different at first glance, however, when we take a closer look at how these toxins function, they are more similar than suggested by the diseases they cause.

Symptoms of Tetanus vs. Symptoms of Botulism

While tetanus causes a body to take a rigid, inflexible state, a very well-described and feared disease since antiquity; botulism reveals itself in limp, uncontrolled muscles, symptoms that mimic those of other diseases, hiding the cause of the disease until the modern age.

Symptoms are strikingly opposite: tetanus is characterized by unrelieved tension or spasticity of the striated muscles and botulism by a limp or flaccid state of the same muscles. In both cases, the muscles can no longer be moved in a coordinated manner, resulting in respiratory paralysis and death.

Tetanus and Botulism Have Similar Basic Origins and Structures

Both diseases can be attributed to toxins created by Clostridia. In general, TeNT is formed by bacteria introduced through injuries such as puncture wounds, placing the bacteria where they can grow in the absence of oxygen. BoNT is also synthesized by bacteria growing under lack of oxygen; however, in contrast to TeNT, botulinum is usually encountered when bacteria multiply and produce toxin in contaminated foods and the toxin is swallowed with the contaminated food. Both toxins enter the bloodstream and are distributed through the body (¹, ², ³, ⁴).

Botulinum and tetanus neurotoxins are both large proteins composed of two parts, a heavy chain, and a light chain. The light chain represents the active component; it is a protease that cleaves peptides regulating exocytosis of neurotransmitters, rendering the nerve unable to communicate. The heavy chain navigates the toxin into target cells and is responsible for transfers through several membranes.

Differences Between Tetanus and Botulism

Although botulinum and tetanus toxins have the same basic structure, tetanus neurotoxin exists solely as a two-part protein neurotoxin; where botulinum toxin is, at least initially, associated with accessory proteins, forming a toxin complex.  This complex can be more than four times larger than the neurotoxin alone (⁵, ⁶).

TeNT is taken up by peripheral cholinergic nerve endings and is transported intraaxonally, retrogradely into the soma of the nerve cell (⁷). It leaves the motor neuron and subsequently enters nerve endings of inhibitory interneurons (⁸, ⁹, ¹⁰, ¹¹). Within the inhibitory neurons, the tetanus enzyme cleaves vesicular VAMP2, inhibiting the release of the transmitters glycine and GABA (¹²). With this action, the fine adjustment of the coordination of motor motion is disturbed. Inhibition is no longer possible so excitatory input is passed unfiltered from the spinal cord to the periphery. Minute peripheral sensory stimuli release a pronounced spasm, the clinical indication of tetanus.  A similar muscle spasm is caused by strychnine, a blocker of glycine receptors.

In addition to this central effect, TeNT also has peripheral effects, splitting VAMP-2 in cholinergic nerve endings, leading to flaccid paralysis. However, this effect is triggered only at about 100-1000 fold higher concentrations, amounts of toxin which are not naturally encountered, so that peripheral effects play no role in clinical tetanus. Peripheral effects can be studied experimentally on isolated nerve-muscle preparations.

It turns out that TeNT largely mimics the effect of BoNTs (¹³). Both TeNT and BoNTs cleave vesicular proteins that trigger fusion of the transmitter-containing vesicles with the plasma membrane. Concentrations of BoNT needed to create paralysis are in general as low as the concentration of TeNT leading to the central effect. The BoNT serotype B not only splits the same protein as TeNT, it even cleaves it in the same place (¹⁴). Clearly, the difference between the action of botulinum and tetanus toxins is the location where the light chain is released and destroys the vesicle docking mechanism.  Transport to the different sites of action is carried out by the heavy chains of these toxins. Surprisingly, BoNT/A and E also enter the soma of motor neurons by retrograde transport and eventually interneurons, where they can trigger central effects (¹⁵, ¹⁶, ¹⁷, ¹⁸, ¹⁹). These effects occur only at high concentrations and are masked by the peripheral paralysis.

Synapses must be actively sending or receiving neurotransmitters to allow endocytosis of both BoNT and TeNT. The reason for this lies in the localization of the receptors for these toxins on the luminal side of the synaptic vesicle. Only after the synaptic vesicle merges and becomes incorporated into the cell membrane do the receptors become accessible to the toxins. However, the dependence of uptake on synaptic activity is only valid if the peripheral effects are involved. Systemic TeNT, which is transported axonally, enters neurons by a different mechanism;  it is endocytosed independent of synaptic activity (10, ²⁰).  TeNT enters vesicles which transport peripheral metabolites via the retrograde route into the soma, for reuse or introduction into other metabolic pathways. TeNT travels on this route as a stowaway.

BoNT serotypes and TeNT are believed to be derived from an ancient toxin that has adapted to different targets in the course of evolution. An adaption allowing the toxin to readily reach a different destination in the nervous system is probably responsible for disguising the toxin.

Contact us with any questions or inquiries regarding our CDMO products and services.

Tetanus Toxin FAQs

• How similar are the amino acid sequences of TeNT and BoNT?
• Why is TeNT not absorbed orally?
• Can BoNT form in poorly perfused human tissue, similar to TeNT?
• Is TeNT like BoNT also synthesized outside of a living organism, for example, in food?
• BoNT is used therapeutically to treat pathological muscle cramping and spasticity. Are there any indications for TeNT, e.g. local paralysis after spinal injuries or stroke?
• Can TeNT like other bacterial toxins be used as a tool in research?
• Is the receptor known for TeNT?
• Is the disease tetanus still a health problem?

Question: How similar are the amino acid sequences of tetanus toxin and botulinum toxin?

Answer: The similarity is about 40-50%, depending on the botulinum toxin serotype.

Question: Why is tetanus toxin not absorbed orally?

Answer: A complex of several proteins protects botulinum toxins from proteolytic degradation in the upper small intestine, this complex is responsible for the oral availability of botulinum toxins. During its further passage in the digestive system, as soon as the pH changes from acid to basic, neurotoxin leaves the complex and is able to enter the circulatory system. Since tetanus toxin has no protective complex proteins, like all other proteins, it is destroyed in the course of the gastrointestinal passage.

Question: Can botulinum toxin form in poorly perfused human tissue, similar to tetanus toxin?

Answer: Clostridium botulinum also grows in poorly perfused tissue injury and can form and release botulinum toxin. Recently, such intoxications have been observed in drug addicts who use injectables contaminated with clostridial spores (²¹). Botulinum toxin is also formed in the intestine of infants when they consume spore-contaminated food, like honey.

Question: Is tetanus toxin like botulinum toxin also synthesized outside of a living organism, for example, in food?

Answer: At least under laboratory conditions, tetanus toxin is produced from bacteria in fermenters. Whether Clostridium tetani naturally produce the toxin under oxygen deficiency outside living organisms is a good question.  Tetanus toxin, without a protective coating, is more vulnerable to the environment than botulinum toxin complex. It does not survive the digestive process when ingested.  Toxin produced outside of a living organism will likely not survive and would not provide a competitive advantage. From the point of view of the organism which uses toxin to secure food and a livable environment, making toxin which is destroyed would be a waste of energy.

Question: botulinum toxin is used therapeutically to treat pathological muscle cramping and spasticity. Are there any indications for tetanus toxin, e.g. local paralysis after spinal injuries or stroke?

Answer: Theoretically one could imagine such applications. In the developed world, however, the population is fully immunized against tetanus toxin, so that injected toxin is immediately neutralized by specific antibodies. A similar situation occurs when antibodies are formed during therapy with botulinum toxin and the botulinum toxin becomes ineffective.

Question: Can tetanus toxin like other bacterial toxins be used as a tool in research?

Answer: Several opportunities are offered by tetanus for research.  Tetanus toxin serves as an aid to the study of axonal transport and has the potential to be used as a carrier for other proteins or substances that are to be channeled into the spinal cord.  Tetanus toxin binds exclusively to neurons and as a result, is an excellent neuronal marker. For this purpose, either the toxin itself or the binding C-fragment can be equipped with a tag like FITC or detected by standard immunology.  Finally, tetanus toxoid is an excellent carrier for antigens used to develop vaccines (²²).

Question: Is the receptor known for tetanus toxin?

Answer: The receptor tetanus toxin is unknown. However, the toxin has two pockets in the binding domain that could recognize different receptors. It is suggested that the receptor responsible for peripheral paralysis is located on the inside of synaptic vesicles like the receptors for the other clostridial neurotoxins and that the receptor that transports the toxin axonally is accessible to the toxin independently of exocytosis. Tetanus toxin like botulinum toxin A is bound to polysialo-gangliosides that reside on the outer side of the plasma membrane of neurons.

Question: Is the disease tetanus still a health problem?

Answer: With the help of immunization of the population against the disease, tetanus occurs only rarely and in unimmunized people. The WHO recommends boost injections every ten years. Tetanus is quite a problem in developing countries. In some states in Africa for example, many infants die from Clostridium tetani infections that occur when umbilical cords are cut with contaminated tools.

References:

1. Popoff MR, Bouvet P 2009 Oct; “Clostridial toxins“ Future Microbiol. 4(8):1021-64. PMID: 19824793

2. Rossetto O, Pirazzini M, Bolognese P, Rigoni M, Montecucco C. 2011 Dec; “An update on the mechanism of action of tetanus and botulinum neurotoxins” Acta Chim Slov. 58(4):702-7 PMID: 24061118

3. Binz T, Rummel A. 2009 Jun; “Cell entry strategy of clostridial neurotoxins” J Neurochem. 109(6):1584-95. PMID: 19457120

4. Pirazzini M, Rossetto O, Eleopra R, Montecucco C 2017 Apr, “Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology“ Pharmacol Rev 69(2):200-235 PMID: 28356439

5. Gu S, Rumpel S, Zhou J, Strotmeier J, Bigalke H, Perry K, Shoemaker CB, Rummel A, Jin 2012 Feb “Botulinum neurotoxin is shielded by NTNHA in an interlocked complex” 24;335(6071):977-81. PMID: 22363010

6. Benefield DA, Dessain SK, Shine N, Ohi MD, Lacy DB 2013 Apr; “Molecular assembly of botulinum neurotoxin progenitor complexes“ Proc Natl Acad Sci USA 110(14):5630-5 PMID: 23509303

7. Erdmann G, Wiegand H, Wellhöner HH. 1975 “Intraaxonal and extraaxonal transport of 125I-tet- anus toxin in early local tetanus” Naunyn Schmiedebergs Arch Pharmacol. 290(4):357- 73 PMID: 53793

8. Surana S, Tosolini AP, Meyer IFG, Fellows AD, Novoselov SS, Schiavo G. 2018 Jun “The travel diaries of tetanus and botulinum neurotoxins” Toxicon 1;147:58-67. PMID: 29031941

9. Bercsenyi K, Giribaldi F, Schiavo G. 2013; “The elusive compass of clostridial neurotoxins: deciding when and where to go?” Curr Top Microbiol Immunol. 364:91-113 PMID: 23239350

10. Lalli G, Bohnert S, Deinhardt K, Verastegui C, Schiavo G. 2003 Sep; “The journey of tetanus and botulinum neurotoxins in neurons” Trends Microbiol. 11(9):431-7. PMID: 13678859

11. Schwab ME, Thoenen H. 1976 Mar “Electron microscopic evidence for a transsynaptic migration of tetanus toxin in spinal cord motoneurons: an autoradiographic and morphometric study” Brain Res. 26;105(2):213-27 PMID: 1260442

12. Brunger AT, Rummel A. 2009 Oct; “Receptor and substrate interactions of clostridial neurotoxins” Toxicon. 54(5):550-6 PMID: 19268493

13. Schmitt A, Dreyer F, John C. 1981; “At least three sequential steps are involved in the tetanus toxin-induced block of neuromuscular transmission” Naunyn Schmiedebergs Arch Pharmacol. 317(4):326-30. PMID: 6119629

14. Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGupta BR, Montecucco C. 1992 Oct “Tetanus and botulinum-B neurotoxins block neurotransmitter release by pro- teolytic cleavage of synaptobrevin” Nature. 29;359(6398):832-5. PMID: 1331807

15. Wiegand H, Erdmann G, Wellhöner HH. 1976; “125I-labelled botulinum A neurotoxin: pharmaco- kinetics in cats after intramuscular injection” Naunyn Schmiedebergs Arch Pharmacol. 292(2):161-5 218

16. Restani L, Giribaldi F, Manich M, Bercsenyi K, Menendez G, Rossetto O, Caleo M, Schiavo G. 2012 Dec; “Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons” PLoS Pathog. 8(12) PMID: 23300443

17. Wiegand H, Wellhöner HH. 1977 Jul; “The action of botulinum A neurotoxin on the inhibition by antidromic stimulation of the lumbar monosynaptic reflex” Naunyn Schmiedebergs Arch Pharmacol. 298(3):235-8. PMID: 895899

18. Restani L, Novelli E, Bottari D, Leone P, Barone I, Galli-Resta L, Strettoi E, Caleo M. 2012 Aug; “Botulinum neurotoxin A impairs neurotransmission following retrograde transynaptic transport” 13(8):1083-9. PMID: 22519601

19. Caleo M, Restani L. 2018 Jun “Direct central nervous system effects of botulinum neurotoxin” Toxicon. 1;147:68-72 PMID: 29111119

20. Bohnert S, Schiavo G. 2005 Dec “Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation” J Biol Chem. 23;280(51):42336-44. PMID: 16236708

21. Gonzales y Tucker RD, Frazee B. 2014 Dec; “View from the front lines: an emergency medicine perspective on clostridial infections in injection drug users” Anaerobe. 30:108-15 PMID: 25230330

22. Aba YT, Cissé L, Abalé AK, Diakité I, Koné D, Kadiané J, Diallo Z, Kra O, Oulaï S, Bissagnéné E. 2016 Aug; “[Neonatal and child tetanus morbidity and mortality in the University hospitals of Abidjan, Côte d’Ivoire (2001-2010)]” Bull Soc Pathol Exot. 109(3):172-9 PMID: 27177642