Review

Authors: Min Dong, Geoffrey Masuyer, Pål Stenmark...

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

Topics: Animals; Botulinum Toxins; Humans; Metalloendopeptidases; Protein Conformation; Protein Engineering; Tetanus Toxin

PubMed: 30388027
DOI: 10.1146/annurev-biochem-013118-111654

Authors: Min-Ju Chang, Morgane Ollivault-Shiflett, Richard Schuman...

Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd (Przedpelski et al. mBio, 2020). Here we describe the production of 8MTT in Gor/Met E. coli, a strain engineered to have an oxidative cytoplasm, allowing for the expression of soluble, disulfide-bonded proteins. The strain was also designed to efficiently cleave N-terminal methionine, the obligatory start amino acid for E. coli expressed proteins. 8MTT was purified as a soluble protein from the cytoplasm in a two-column protocol to > 99 % purity, yielding 0.5 g of purified 8MTT/liter of fermentation broth with low endotoxin contamination, and antigenic purity of 3500 Lf/mg protein nitrogen. Mouse immunizations showed 8MTT to be an immunogenic vaccine and effective as a carrier protein for peptide and polysaccharide conjugates. These studies validate 8MTT as commercially viable and, unlike the heterogenous tetanus toxoid, a uniform carrier protein for conjugate vaccines. The development of a recombinant, genetically detoxified toxin produced in E. coli aligns the tetanus vaccine with modern manufacturing, regulatory, standardization, and safety requirements.

Topics: Animals; Antibodies, Bacterial; Carrier Proteins; Escherichia coli; Mice; Tetanus; Tetanus Toxin; Tetanus Toxoid; Vaccines, Conjugate

PubMed: 35871872
DOI: 10.1016/j.vaccine.2022.07.011

Authors: Sunaina Surana, David Villarroel-Campos, Elena R Rhymes...

Tetanus neurotoxin (TeNT) causes spastic paralysis by inhibiting neurotransmission in spinal inhibitory interneurons. TeNT binds to the neuromuscular junction, leading to its internalisation into motor neurons and subsequent transcytosis into interneurons. While the extracellular matrix proteins nidogens are essential for TeNT binding, the molecular composition of its receptor complex remains unclear. Here, we show that the receptor-type protein tyrosine phosphatases LAR and PTPRδ interact with the nidogen-TeNT complex, enabling its neuronal uptake. Binding of LAR and PTPRδ to the toxin complex is mediated by their immunoglobulin and fibronectin III domains, which we harnessed to inhibit TeNT entry into motor neurons and protect mice from TeNT-induced paralysis. This function of LAR is independent of its role in regulating TrkB receptor activity, which augments axonal transport of TeNT. These findings reveal a multi-subunit receptor complex for TeNT and demonstrate a novel trafficking route for extracellular matrix proteins. Our study offers potential new avenues for developing therapeutics to prevent tetanus and dissecting the mechanisms controlling the targeting of physiological ligands to long-distance axonal transport in the nervous system.

Topics: Animals; Mice; Tetanus Toxin; Motor Neurons; Membrane Glycoproteins; Humans; Cell Adhesion Molecules; Protein Binding; Receptor, trkB; Axonal Transport; Receptor-Like Protein Tyrosine Phosphatases, Class 2

PubMed: 38977849
DOI: 10.1038/s44318-024-00164-8

Review

Authors: B Bizzini

Topics: Animals; Cell Membrane; Cells, Cultured; Central Nervous System; Chemical Phenomena; Chemistry; Clostridium tetani; Gangliosides; Humans; Models, Chemical; Receptors, Drug; Tetanus; Tetanus Toxin

PubMed: 390355
DOI: 10.1128/mr.43.2.224-240.1979

Review

Authors: Janne M Toivonen, Sara Oliván, Rosario Osta...

In many neurological disorders strategies for a specific delivery of a biological activity from the periphery to the central nervous system (CNS) remains a considerable challenge for successful therapy. Reporter assays have established that the non-toxic C-fragment of tetanus toxin (TTC), provided either as protein or encoded by non-viral naked DNA plasmid, binds pre-synaptic motor neuron terminals and can facilitate the retrograde axonal transport of desired therapeutic molecules to the CNS. Alleviated symptoms in animal models of neurological diseases upon delivery of therapeutic molecules offer a hopeful prospect for TTC therapy. This review focuses on what has been learned on TTC-mediated neuronal targeting, and discusses the recent discovery that, instead of being merely a carrier molecule, TTC itself may well harbor neuroprotective properties.

Topics: Animals; Axonal Transport; Disease Models, Animal; Gene Targeting; Genetic Therapy; Humans; Molecular Targeted Therapy; Motor Neuron Disease; Motor Neurons; Neural Pathways; Neurodegenerative Diseases; Neuromuscular Junction; Neuroprotective Agents; Peptide Fragments; Presynaptic Terminals; Tetanus Toxin

PubMed: 22069568
DOI: 10.3390/toxins2112622

Authors: Christopher C Matthews, Paul S Fishman, George F Wittenberg...

INTRODUCTION

Skeletal muscles that are under the influence of tetanus toxin show an exaggerated reflex response to stretch. We examined which changes in the stretch reflex may underlie the exaggerated response.

METHODS

H-reflexes were obtained from the tibialis anterior (TA) and flexor digitorum brevis (FDB) muscles in rats 7 days after intramuscular injection of tetanus toxin into the TA.

RESULTS

We found effects of the toxin on the threshold, amplitude, and duration of H-waves from the TA. The toxin inhibited rate-dependent depression in the FDB between the stimulation frequencies of 0.5–50 HZ and when a conditioning magnetic stimulus applied to the brain preceded a test electrical stimulus delivered to the plantar nerve.

CONCLUSIONS

Tetanus toxin increased the amplitude of the Hwave and reduced the normal depression of H-wave amplitude that is associated with closely timed stimuli, two phenomena that could contribute to hyperactivity of the stretch reflex.

Topics: Animals; Electric Stimulation; Female; H-Reflex; Muscle, Skeletal; Rats; Rats, Sprague-Dawley; Reflex, Stretch; Tetanus Toxin

PubMed: 24772492
DOI: 10.1002/mus.23938

Authors: Yasuhiro Watanabe, Takashi Matsuba, Mami Nakanishi...

BACKGROUND

Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments.

RESULTS

We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport.

CONCLUSIONS

These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.

Topics: Animals; Axonal Transport; COS Cells; Cell Line; Chlorocebus aethiops; Cytoprotection; Escherichia coli; Mice, Inbred C57BL; Nervous System Diseases; Neurons; Peptide Fragments; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Tetanus Toxin

PubMed: 29890980
DOI: 10.1186/s12896-018-0452-z

Authors: C L Thwaites, J J Farrar

Topics: Global Health; Humans; Tetanus; Tetanus Toxin

PubMed: 12531822
DOI: 10.1136/bmj.326.7381.117

Authors: Mehdi Yousefi, Vahid Younesi, Ali Ahmad Bayat...

Tetanus is a highly fatal disease caused by tetanus neurotoxin (TeNT) and remains a major threat to human and animal health, despite preventive strategies. TeNT is composed of heavy and light chain linked by a disulfide bond. The antibody response to TeNT is polyclonal and directed to multiple epitopes within both the light and heavy chains, leading to toxin neutralization. This study was undertaken to localize and compare neutralizing epitopes recognized by human and mouse TeNT-specific antibodies at a clonal level. In the present study, 22 murine hybridoma clones and 50 human lymphoblastoid cell lines secreting monoclonal antibodies (mAb) were generated against TeNT. The specificity of these mAb was determined using different recombinant fragments of tetanus toxin. Moreover, this study investigated the in vitro toxin neutralizing activity of these mAb by a ganglioside GT1b assay. The results showed that tetanus toxoid immunization in humans and BALB/c mice induced a vigorous humoral immune response against different fragments of TeNT, particularly the carboxyl-terminal fragment of the heavy chain (known as fragment C). The fragment C-specific human and mouse mAb could largely neutralize TeNT. However, while all fragment C-specific human mAb reacted with the carboxyl-terminal part of this fragment (H(CC)), the majority of the mouse mAb failed to recognize this region. These results suggested that fragment C is the major target for the TeNT neutralizing antibodies, although different epitopes seem to be targeted by human and mouse antibodies.

Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal, Murine-Derived; Antibodies, Neutralizing; Antibody Formation; Female; Humans; Male; Mice; Species Specificity; Tetanus Toxin

PubMed: 25990600
DOI: 10.3109/1547691X.2015.1046572

Review

Authors: Ornella Rossetto, Cesare Montecucco

Tetanus and botulinum neurotoxins are the most poisonous substances known, so much so as to be considered for a possible terrorist use. At the same time, botulinum neurotoxin type A1 is successfully used to treat a variety of human syndromes characterized by hyperactive cholinergic nerve terminals. The extreme toxicity of these neurotoxins is due to their neurospecificity and to their metalloprotease activity, which results in the deadly paralysis of tetanus and botulism. Recently, many novel botulinum neurotoxins and some botulinum-like toxins have been discovered. This large number of toxins differs in terms of toxicity and biological activity, providing a potential goldmine for novel therapeutics and for new molecular tools to dissect vesicular trafficking, fusion, and exocytosis. The scattered data on toxicity present in the literature require a systematic organization to be usable by scientists and clinicians. We have assembled here the data available in the literature on the toxicity of these toxins in different animal species. The internal comparison of these data provides insights on the biological activity of these toxins.

Topics: Animals; Botulinum Toxins; Humans; Lethal Dose 50; Neurotoxins; Tetanus Toxin

PubMed: 31771110
DOI: 10.3390/toxins11120686