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 BotulismHave 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.
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.
Question: How similar are the amino acid sequences of TeNT and BoNT?
Answer: The similarity is about 40-50%, depending on the BoNT serotype.
Question: Why is TeNT not absorbed orally?
Answer: A complex of several proteins protects BoNTs 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. SinceTeNT has no protective complex proteins, like all other proteins, it is destroyed in the course of the gastrointestinal passage.
Question: Can BoNT form in poorly perfused human tissue, similar to TeNT?
Answer: Clostridium botulinum also grows in poorly perfused tissue injury and can form and release BoNT. Recently, such intoxications have been observed in drug addicts who use injectables contaminated with clostridial spores (21). BoNT is also formed in the intestine of infants when they consume spore-contaminated food, like honey.
Question: Is TeNT like BoNT also synthesized outside of a living organism, for example, in food?
Answer: At least under laboratory conditions, TeNT 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: 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?
Answer: Theoretically one could imagine such applications. In the developed world, however, the population is fully immunized against TeNT, so that injected toxin is immediately neutralized by specific antibodies. A similar situation occurs when antibodies are formed during therapy with BoNT and the BoNT becomes ineffective.
Question: Can TeNT like other bacterial toxins be used as a tool in research?
Answer: Several opportunities are offered by tetanus for research. TeNT 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. TeNT 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 TeNT?
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. TeNT like BoNT/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.
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
Binz T, Rummel A. 2009 Jun; “Cell entry strategy of clostridial neurotoxins” J Neurochem. 109(6):1584-95. PMID: 19457120
Pirazzini M, Rossetto O, Eleopra R, Montecucco C 2017 Apr, “Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology“ Pharmacol Rev 69(2):200-235 PMID: 28356439
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
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
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
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
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
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
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
Brunger AT, Rummel A. 2009 Oct; “Receptor and substrate interactions of clostridial neurotoxins” Toxicon. 54(5):550-6 PMID: 19268493
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
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
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
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
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
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
Caleo M, Restani L. 2018 Jun “Direct central nervous system effects of botulinum neurotoxin” Toxicon. 1;147:68-72 PMID: 29111119
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
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
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
By: Mary N. Wessling, Ph.D. ELS
List Biological Laboratories’ (List Labs) catalog of products is related to furthering research in human health and preventing disease, most commonly as the starting materials for vaccine research & development or production around the world. Vaccines are mainly identified for their capacity to prevent diseases that the body’s innate defensive mechanisms (the skin and specialized cells in the blood, for example) can’t resist unaided. However, there are many other uses for these purified materials in medical research, and you will likely encounter wording on our website that is not part of everyday vocabulary for non-scientists. This article is intended to provide a basic understanding of some of the more frequently used terms and aid you in selecting the products most essential to your projects.
Toxin vs. Toxoid
For starters, what is the difference between “toxin” and “toxoid”. Broadly defined, anything that can cause harm to an organism is a toxin. However, for List Labs’ products and in biological usage, a toxin refers to a bacterial or viral product that has harmful effects when it enters the body (List Labs’ toxins are in a highly purified form). A toxoid is a chemically altered toxin that has reduced or no toxicity and is used for its remaining antigenic activity, which can stimulate an immune response.
Take, for example, cholera, a disease produced by Vibrio cholerae bacteria, possibly through contact with body fluids from a person ill with cholera or through contaminated water supply. Cholera causes severe diarrhea, and untreated, it can be fatal. However, the purified List Labs’ cholera toxin by chemical modification becomes a toxoid that lacks toxic activity but retains structures that make it useful for immunization of research animals or stimulation of immune cells in vitro.
How do Toxoids Impact the Immune Process?
To understand how some List Labs products work, an overview of the immune process is helpful. During the course of a day, we frequently touch, ingest, or breathe in something that has potential to harm the body. Our cells react to this invader: is this a threat, or not, and if so, how serious is the threat?
What is the Innate Immune Response?
The innate immune response is the first order of defense in the immune process. There are many different cell types in our body. Some of these cells are equipped by their structural and biochemical components to destroy dangerous microbial invaders–pathogens–quickly. The inflammation that we experience from minor infections is often a sign of this process as cells from the blood destroy the pathogen. This happens quickly, within hours.
What is the Adaptive Immune Response?
Another cellular response system requires a longer time to react to the threat. These cells react by changing from an inactive form to one that will start a more complex defensive process: this is the second step, the adaptive immune response. There are two different classes of cells that comprise the adaptive immune response; they differ by the structures that give them their ability to bind antigens– the invading bacteria and viruses. Both these cells are called lymphocytes; individually, they are the B-lymphocytes (B-cells) and T-lymphocytes (T-cells). Both originate from stem cells in the bone marrow; B and T refer to the place in the body where they mature. T-cells mature in the thymus into several subclasses of T-cells that circulate in the blood and lymph. “Killer” T-cells recognize foreign antigens on cell surfaces (e.g. from viral infection or malignancy). “Helper” T-cells induce B-cells to produce antibodies. “Suppressor” T-cells dampen or regulate the immune response to prevent over-reaction. B-cells mature in the bone marrow and migrate to secondary lymphoid tissues (e.g. spleen and lymph nodes). When they encounter foreign antigens and/or helper T-cells, they are stimulated to divide and expand clonally to produce antibodies and differentiate into plasma cells.
What is Immune Memory?
After the B and T-lymphocytes react to an antigen, two results are possible. The first, and desirable result, is that the invader is identified and defeated, leaving behind what might be called its criminal record: immune memory. When the antigen comes creeping back in the future, the adaptive immune system recognizes it and attacks. The second possibility is an over-reaction and lack of cessation of the adaptive immune process that is harmful to the body: an autoimmunecondition.
Antigens, Epitopes and Vaccines
Where do vaccines come into this process? An antigen is a substance that causes the body to mount an immune response against it. Antigens include toxins, bacteria, viruses, or other substances that the body recognizes as foreign or not “self”. Vaccines have structural features similar to structures of the toxin or invading pathogen that can elicit adaptive immunity.
An epitope is a specific molecular region on the surface of an antigen, typically one of many on the antigen, that elicits an immune response and is capable of binding with the specific antibody produced by the response. A toxin has many epitopes that can be recognized by the immune response. The epitopes that are required for toxicity have been altered chemically in toxoids or by specific genetic mutations in inactive mutants; however, many epitopes are retained and can stimulate an adaptive or memory immune response that will be effective against the toxin.
Toxins and Toxoid Products for Research
Below is a list of toxin and toxoid or inactive mutant pairs of products available to support your research.
Toxin and product numbers
Botulinum Neurotoxin Type A from Clostridiumbotulinum – 130A, 130B, 9130A
Tetanus Toxin’s Use as a Protein Carrier and Antigen
Tetanus toxin (TT) is the major virulence factor of the Gram-positive bacterium Clostridium tetani. Infection with this bacterium in unvaccinated persons produces muscle spasms by binding to nerve endings and moving throughout the nervous system in a specific way. Eventually, almost total paralysis results. The deactivated toxin is the basis for a vaccine, which can even be administered to pregnant women, usually as part of a combination vaccine also aimed at preventing neonatal pertussis.1Worldwide, the mortality of infection among unvaccinated persons reaches 10%.2 It is the binding specificity of this dangerous toxin that makes it valuable as a research material. List Biological Laboratories (List Labs) offers inactivated tetanus toxin and six related products, used in intricate and fascinating ways in research as a model antigen and protein carrier.
List Labs’ Tetanus Toxoid in Immunosupression Research
Existing antibody treatments for rheumatic arthritis (RA), for example doses of rituximab every six months, suppress autoreactive B cells by killing them. The effects of the treatment fade over the 6-month period; this increases the inherent risk of infection and progressive multifocal leukoencephalopathy. Chu et al studied a treatment that characterized B-cell immunosuppression by an engineered antibody; List Labs’ tetanus toxoid (TTd)
was used in an elegant series of explorations of the mechanism of action of an engineered antibody (XmAb581) that enhanced the action of a B-cell antigen receptor complex currently under clinical development for treatment of RA, and enhanced both its safety and efficacy.3
Tetanus Toxoid in B Cell-Driven Autoimmune Disease Research
Prompted by the lower prevalence of these diseases in children who lived near farm animals and in unhygienic environments, Iwasaki et al studied the key role of intestinal infection in development of allergic respiratory disease in children. Their study used List Labs’ TTd antigen
was used in a study that challenged an accepted mechanism for cell death in injured human retinal ganglion cells; Li Y et al showed that dysregulation of mobile zinc is to blame. In anesthetized animals, they used TTd to cleave the synaptic vesicle protein and then injected the zinc formulation. Fluorescent images showed that there was a rapid accumulation of Zn2+ in amacrine cell processes after optic nerve injury.6 Investigating yet another problem that affects injured persons, i.e., the necessity to keep an injured limb immobile, which results in muscle atrophy, Matthews et al reported that inactivity can result in 20% to 30% atrophy despite the use of exercise-based or neuromuscular electronic stimulation. They injected either saline or a very dilute solution of List Labs’ TT
Finally, animal studies are used to evaluate the efficacy of pharmaceutical and other products for human use; although there are stringent conditions that assure ethical treatment of animals, the process is often time-inefficient, inaccurate, and costly. Temann et al explored using precision-cut lung slices (PCLS) from lungs of donors that were not suitable for use in transplantation as an alternative to animal studies. They evaluated a culture system using PCLS stimulated by List Labs’ TTd
The studies we cite here are only a small sample of what can be accomplished using List Labs’ TT and related products. We invite you to visit our citations page to explore how TT and our other products can augment your experimental design.
da Silva Antunes R, Paul S, Sidney J, et al. Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses. PloS One. 2017;12(1):e0169086. PMID: 28081174
Chu SY, Yeter K, Kotha R, et al. Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcgamma receptor IIb inhibitory receptor. Arthritis & Rheumatology (Hoboken, NJ). 2014;66(5):1153-1164. PMID: 24782179
Klose D, Saunders U, Barth S, Fischer R, Jacobi AM, Nachreiner T. Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen. BMC Biotechnology. 2016;16:18. PMCID: PMC4756516
Iwasaki J, Chai LY, Khoo SK, et al. Lower anti-echovirus antibody responses in children presenting to hospital with asthma exacerbations. Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology. 2015;45(10):1523-1530. PMID: 25640320
Li Y, Andereggen L, Yuki K, et al. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration. Proceedings of the National Academy of Sciences of the United States of America. 2017;114(2):E209-e218. PMCID: PMC5240690
Matthews CC, Lovering RM, Bowen TG, Fishman PS. Tetanus toxin preserves skeletal muscle contractile force and size during limb immobilization. Muscle & Nerve. 2014;50(5):759-766. PMID: 24590678
Temann A, Golovina T, Neuhaus V, et al. Evaluation of inflammatory and immune responses in long-term cultured human precision-cut lung slices. Human Vaccines & Immunotherapeutics. 2017;13(2):351-358. PMID: 27929748
By: Dom C. Ouano, Marketing Coordinator
Since List Labs introduced products in the Tetanus Toxin family more than 25 years ago, interest and knowledge in this field has multiplied. Tetanus Toxin C fragment, the non-toxic C-terminal domain of the heavy chain, is retrogradely transported to the central nervous system and is useful as a neuronal tracer and a biological carrier. It is also reported to have neuroprotective effects in mice, providing protection against methamphetamine induced neurotoxicity and motor impairment. Tetanus Toxin is used in an animal model of temporal lobe epilepsy, and Tetanus Toxoid is a recall (memory) antigen for activation of peripheral blood mononuclear cells (PBMC). Tetanus Toxoid is used as a carrier protein for glycoconjugate vaccines, and acts as a vaccine adjuvant stimulating protective immune responses.
List Labs offers Tetanus Toxin C-Fragment from Clostridium tetani in 10ug vials (Product #193). Tetanus Toxin C-Fragment from Clostridium tetani, FITC Conjugate is available in 10ug vials (Product #196A). Tetanus Toxin from Clostridium tetani (Product #190) is available in either 25ug (#190A) or 100ug (#190B) vials. Tetanus Toxoid from Clostridium tetani (Product #191) is available in either 25ug (#191A) or 100ug (#191B) vials. Learn about our entire family of Tetanus products here.