Information

Persistency of botulinum toxin in environment


There is information about decontamination times here on pages 6 and 7. My specific question is how persistent botulinum toxin is in a natural environment, or alternatively, what is the half life of this toxin?


History, Science and Methods

Abstract

Clostridium botulinum is a heterogeneous species containing four phylogenetically and physiologically distinct bacteria that share the common feature of forming the botulinum neurotoxin. Some strains of Clostridium baratii and Clostridium butyricum also produce botulinum neurotoxins. There are seven botulinum neurotoxins (types A–G). These are the most potent toxins known (as little as 30 ng is sufficient to cause illness and possibly death), and are responsible for botulism, a severe and often fatal neuroparalytic intoxication. The extreme severity of this disease requires that regulators and industry remain vigilant to minimize the foodborne botulism risk.


Introduction

The species comprises multiple highly heterogeneous strains of rod-shaped anaerobic spore-forming bacteria, which are categorized into four groups (Groups I–IV) based on genomic relatedness. All C. botulinum strains produce botulinum toxin, which paralyzes animals by inhibiting acetylcholine release from synaptic vesicles at neuromuscular junctions. This toxin is classified into eight serotypes designated A–H (Collins and East, 1998 Barash and Arnon, 2014), of which A, B, E, and F are shown toxic to humans. Botulinum toxin-producing bacteria are divided into six groups: C. botulinum Groups I–IV as well as some strains of C. baratii and C. butyricum (Peck, 2009). Group I includes the proteolytic C. botulinum strains that produce botulinum toxin serotypes A, B, and F. Group II comprises non-proteolytic strains that produce toxin serotypes B, E, and F. The strains in Group III produce serotypes C and D, or mosaic C/D toxins. Group VI strains, referred to as C. argentinense (Suen et al., 1988), produce toxin serotype G. Among the other species, C. butyricum produces botulinum toxin serotype E and C. baratii produces serotype F (Hill et al., 2009).

Botulinum toxin genes exhibit remarkably variable organization. They can be chromosomally localized or localized on plasmids or phages (serotypes C and D). Serotype B transcription can occur through both genome-encoded and plasmid-encoded toxin gene clusters (Franciosa et al., 2009). Genome comparisons have revealed evidence of toxin cluster evolution through horizontal gene transfer, site-specific insertion, and recombination, and genomic analysis has supported the historic group classifications (Hill and Smith, 2013 Stringer et al., 2013). Thus, the factors affecting pathogenicity are apparently subjected to a higher evolutionary rate than the core genomes, allowing for fast environmental adaptation of the pathogen.

The ecology and properties are similar enough among Groups I–IV that it remains meaningful to discuss C. botulinum in the environment as a single group. C. botulinum spores persist in soils and aquatic sediments for decades, and propagate by predator-dependent disease transmission. Upon entering the food webs of animals, C. botulinum toxins may intoxicate and kill the animal, or infect and proliferate and kill the prey. Saprophytic utilization of the prey via enzymes, including proteases and chitinases, makes nutrients available for massive spore and toxin production. Neurotoxin gene expression and toxin complex formation reportedly occur in the late exponential growth phase and the early stationary phase (Bradshaw et al., 2004 Kouguchi et al., 2006 Artin et al., 2008 Cooksley et al., 2010), and toxin production and sporulation seem to be co-regulated (Cooksley et al., 2010).

It appears that contaminated soils and sediments are primary environments for spores and serve as an incubation area, from which the pathogens may be mobilized (Long and Tauscher, 2006). C. botulinum is detected in, or may be associated with, various organisms that are not affected by the toxins—such as algae, plants, and invertebrates (Quortrup and Holt, 1941 Duncan and Jensen, 1976 Bohnel, 2002). Fish are carriers of C. botulinum, but botulism outbreaks in fish populations may lead to death on a large scale (Yule et al., 2006 Hannett et al., 2011). Avian botulism caused by C. botulinum type C, mosaic C/D, or E is a common cause of death among waterfowl (Skulberg and Holt, 1987 Friend, 2002 Takeda et al., 2005 Lafrancois et al., 2011 Vidal et al., 2013). Unpredictable outbreaks with variable losses have been reported worldwide (Friend, 2002 Babinszky et al., 2008 Shin et al., 2010 Vidal et al., 2013). In recent years, large outbreaks in the Great Lakes, with high mortalities among fish and birds, have been well documented and analyzed (Perez-Fuentetaja et al., 2006, 2011 Lafrancois et al., 2011 Chun et al., 2013). In this review, we discuss factors related to botulism outbreaks in natural environments.


Inhibition of Botulinum Toxin Formation in Bacon by Acid Development

N. TANAKA, E. TRAISMAN, M. H. LEE, R. G. CASSENS, E. M. FOSTER Inhibition of Botulinum Toxin Formation in Bacon by Acid Development. J Food Prot 1 June 1980 43 (6): 450–457. doi: https://doi.org/10.4315/0362-028X-43.6.450

Lactobacillus plantarum, as a producer of lactic acid, and sucrose as a fermentable carbohydrate were evaluated for use in lowering the amount of or eliminating sodium nitrite in bacon. This work was limited to effect on antibotulinal properties. Organoleptic effects were not considered. Slices of bacon were inoculated with spores of Clostridium botulinum types A and B with or without simultaneous inoculation with a culture of L. plantarum, vacuum-packaged and incubated at 27 C. Samples were taken after various periods of incubation and assayed for botulinal toxin. We found that (a) sodium nitrite alone, at 120 ppm, did not give bacon extended protection against development of botulinum toxin if a fermentable carbon source (sucrose in these instances) was not present (b) without added lactic acid bacteria, the effectiveness of 120 ppm of sodium nitrite plus sugar was variable and depended upon growth of naturally contaminating bacteria and (c) lactic acid bacteria with an adequate amount of sucrose gave good protection against development of botulinal toxin. Upon temperature abuse, acid was produced and growth of C. botulinum was inhibited. Because the protective properties against development of botulinal toxin in the sugar-lactic acid bacteria system were not dependent on the presence of nitrite, nitrite can be lowered to the level necessary to make organoleptically acceptable products without sacrificing safety, thus less nitrosamine formation may be achieved.


Popoff, M. R. & Bouvet, P. Genetic characteristics of toxigenic Clostridia and toxin gene evolution. Toxicon 75, 63–89 (2013).

Johnson, E. A. & Montecucco, C. in Handbook of Clinical Neurology Vol. 91, 333–368 (ed. Engel, A. G.) (Elsevier, 2008).

Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis. Physiol. Rev. 80, 717–766 (2000).

Cherington, M. Clinical spectrum of botulism. Muscle Nerve 21, 701–710 (1998).

Centers for Disease Control and Prevention, Department of Health and Human Services. Possession, use, and transfer of select agents and toxins biennial review. Final rule. Fed. Regist. 77, 61083–61115 (2012).

Arnon, S. S. et al. Botulinum toxin as a biological weapon: medical and public health management. J. Am. Med. Ass. 285, 1059–1070 (2001).

Lim, E. C. & Seet, R. C. Use of botulinum toxin in the neurology clinic. Nature Rev. Neurol. 6, 624–636 (2010).

Smith, L. D. S. & Sugiyama, H. Botulism: the Organism, its Toxins, the Disease (Charles C. Thomas Publisher, 1988).

Hill, K. K. & Smith, T. J. Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes. Curr. Top. Microbiol. Immunol. 364, 1–20 (2013).

Rocke, E. T. & Samuel, M. D. Water and sediment characteristics associated with avian botulism outbreaks in wetlands. J. Wildl. Management 63, 1249–1260 (1999).

Aureli, P. et al. Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy. J. Infect. Dis. 154, 207–211 (1986). This is the first report of botulism caused by a clostridial species other than C. botulinum.

Koepke, R., Sobel, J. & Arnon, S. S. Global occurrence of infant botulism, 1976–2006. Pediatrics 122, e73–e82 (2008).

Simpson, L. L. The life history of a botulinum toxin molecule. Toxicon 68, 40–59 (2013).

Wenham, T. N. Botulism: a rare complication of injecting drug use. Emerg. Med. J. 25, 55–56 (2008).

Chertow, D. S. et al. Botulism in 4 adults following cosmetic injections with an unlicensed, highly concentrated botulinum preparation. J. Am. Med. Ass. 296, 2476–2479 (2006).

Dover, N., Barash, J. R., Hill, K. K., Xie, G. & Arnon, S. S. Molecular characterization of a novel botulinum neurotoxin type H gene. J. Infect. Dis. 209, 192–202 (2014).

Lacy, D. B., Tepp, W., Cohen, A. C., DasGupta, B. R. & Stevens, R. C. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nature Struct. Biol. 5, 898–902 (1998). This study reports the first crystal structure of a BoNT and provides the molecular basis for understanding the mechanism of neuron intoxication.

Swaminathan, S. & Eswaramoorthy, S. Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B. Nature Struct. Biol. 7, 693–699 (2000).

Kumaran, D. et al. Domain organization in Clostridium botulinum neurotoxin type E is unique: its implication in faster translocation. J. Mol. Biol. 386, 233–245 (2009).

Gu, S. et al. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science 335, 977–981 (2012). This study reports the unexpected finding that NTNHA adopts a similar fold to BoNT and that together, the two proteins form an interlocked complex, which suggests that NTNHA stabilizes BoNT and protects the toxin against proteolytic cleavage.

Bonventre, P. F. Absorption of botulinal toxin from the gastrointestinal tract. Rev. Infect. Dis. 1, 663–667 (1979).

Ohishi, I. & Sakaguchi, G. Oral toxicities of Clostridium botulinum type C and D toxins of different molecular sizes. Infect. Immun. 28, 303–309 (1980).

Lee, K. et al. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoS Pathog. 9, e1003690 (2013).

Benefield, D. A., Dessain, S. K., Shine, N., Ohi, M. D. & Lacy, D. B. Molecular assembly of botulinum neurotoxin progenitor complexes. Proc. Natl Acad. Sci. USA 110, 5630–5635 (2013).

Sugawara, Y. et al. Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin. J. Cell Biol. 189, 691–700 (2010).

Fujinaga, Y., Sugawara, Y. & Matsumura, T. Uptake of botulinum neurotoxin in the intestine. Curr. Top. Microbiol. Immunol. 364, 45–59 (2013).

Couesnon, A., Molgo, J., Connan, C. & Popoff, M. R. Preferential entry of botulinum neurotoxin A H domain through intestinal crypt cells and targeting to cholinergic neurons of the mouse intestine. PLoS Pathog. 8, e1002583 (2012).

Maksymowych, A. B. et al. Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade. Infect. Immun. 67, 4708–4712 (1999).

Restani, L. et al. Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons. PLoS Pathog. 8, e1003087 (2012).

Sheth, A. N. et al. International outbreak of severe botulism with prolonged toxemia caused by commercial carrot juice. Clin. Infect. Dis. 47, 1245–1251 (2008).

Fagan, R. P., McLaughlin, J. B. & Middaugh, J. P. Persistence of botulinum toxin in patients' serum: Alaska, 1959–2007. J. Infect. Dis. 199, 1029–1031 (2009).

Dolly, J. O., Black, J., Williams, R. S. & Melling, J. Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization. Nature 307, 457–460 (1984). This study provides the first evidence that BoNTs bind specifically to the presynaptic membrane before entering the nerve terminal.

Montecucco, C. How do tetanus and botulinum toxins bind to neuronal membranes? Trends Biochem. Sci. 11, 314–317 (1986). This paper proposes that dual receptor binding could account for the high specificity and affinity of tetanus toxin and BoNTs for the presynaptic membrane.

Rummel, A. Double receptor anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity. Curr. Top. Microbiol. Immunol. 364, 61–90 (2013).

Chai, Q. et al. Structural basis of cell surface receptor recognition by botulinum neurotoxin B. Nature 444, 1096–1100 (2006).

Jin, R., Rummel, A., Binz, T. & Brunger, A. T. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity. Nature 444, 1092–1095 (2006).

Berntsson, R. P., Peng, L., Dong, M. & Stenmark, P. Structure of dual receptor binding to botulinum neurotoxin B. Nature Commun. 4, 2058 (2013). References 35, 36 and 37 describe the crystallographic structure of BoNT/B in complex with both its protein receptor and glycolipid receptor, which provides experimental evidence for the dual receptor binding model.

Montecucco, C., Rossetto, O. & Schiavo, G. Presynaptic receptor arrays for clostridial neurotoxins. Trends Microbiol. 12, 442–446 (2004).

Muraro, L., Tosatto, S., Motterlini, L., Rossetto, O. & Montecucco, C. The N-terminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane. Biochem. Biophys. Res. Commun. 380, 76–80 (2009).

Zhang, Y. et al. Structural insights into the functional role of the Hn sub-domain of the receptor-binding domain of the botulinum neurotoxin mosaic serotype C/D. Biochimie 95, 1379–1385 (2013).

Van Heyningen, W. E. Tentative identification of the tetanus toxin receptor in nervous tissue. J. Gen. Microbiol. 20, 310–320 (1959). This paper provides the first experimental evidence that a ganglioside is involved in the neurospecific binding of a clostridial neurotoxin.

Simpson, L. L. & Rapport, M. M. The binding of botulinum toxin to membrane lipids: sphingolipids, steroids and fatty acids. J. Neurochem. 18, 1751–1759 (1971).

Simons, K. & Toomre, D. Lipid rafts and signal transduction. Nature Rev. Mol. Cell Biol. 1, 31–39 (2000).

Prinetti, A., Loberto, N., Chigorno, V. & Sonnino, S. Glycosphingolipid behaviour in complex membranes. Biochim. Biophys. Acta 1788, 184–193 (2009).

Chiba, A., Kusunoki, S., Shimizu, T. & Kanazawa, I. Serum IgG antibody to ganglioside GQ1b is a possible marker of Miller Fisher syndrome. Ann. Neurol. 31, 677–679 (1992).

Bullens, R. W. et al. Complex gangliosides at the neuromuscular junction are membrane receptors for autoantibodies and botulinum neurotoxin but redundant for normal synaptic function. J. Neurosci. 22, 6876–6884 (2002).

Fogolari, F., Tosatto, S. C., Muraro, L. & Montecucco, C. Electric dipole reorientation in the interaction of botulinum neurotoxins with neuronal membranes. FEBS Lett. 583, 2321–2325 (2009).

Black, J. D. & Dolly, J. O. Interaction of 125 I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis. J. Cell Biol. 103, 535–544 (1986).

Strotmeier, J. et al. Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner. Biochem. J. 431, 207–216 (2010).

Karalewitz, A. P., Fu, Z., Baldwin, M. R., Kim, J. J. & Barbieri, J. T. Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry. J. Biol. Chem. 287, 40806–40816 (2012).

Strotmeier, J. et al. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites. Mol. Microbiol. 81, 143–156 (2011).

Pirazzini, M., Rossetto, O., Bolognese, P., Shone, C. C. & Montecucco, C. Double anchorage to the membrane and intact inter-chain disulfide bond are required for the low pH induced entry of tetanus and botulinum neurotoxins into neurons. Cell. Microbiol. 13, 1731–1743 (2011).

Kitamura, M., Takamiya, K., Aizawa, S. & Furukawa, K. Gangliosides are the binding substances in neural cells for tetanus and botulinum toxins in mice. Biochim. Biophys. Acta 1441, 1–3 (1999).

Yowler, B. C., Kensinger, R. D. & Schengrund, C. L. Botulinum neurotoxin A activity is dependent upon the presence of specific gangliosides in neuroblastoma cells expressing synaptotagmin I. J. Biol. Chem. 277, 32815–32819 (2002).

Jacky, B. P. S. et al. Identification of fibroblast growth factor receptor 3 (FGFR3) as a protein receptor for botulinum neurotoxin serotype A (BoNT/A). PLoS Pathog. 9, e1003369 (2013).

Nishiki, T. et al. Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes. J. Biol. Chem. 269, 10498–10503 (1994). This study is the first to identify a synaptic vesicle protein receptor for a BoNT by showing that BoNT/B binds to Syt.

Dong, M. et al. Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells. J. Cell. Biol. 162, 1293–1303 (2003).

Rummel, A. et al. Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept. Proc. Natl Acad. Sci. USA 104, 359–364 (2007).

Peng, L. et al. Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D–C and G toxins. J. Cell Sci. 125, 3233–3242 (2012).

Berntsson, R. P., Peng, L., Svensson, L. M., Dong, M. & Stenmark, P. Crystal structures of botulinum neurotoxin dc in complex with its protein receptors synaptotagmin I and II. Structure 21, 1602–1611 (2013).

Dong, M. et al. SV2 is the protein receptor for botulinum neurotoxin A. Science. 312, 592–596 (2006).

Dong, M. et al. Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons. Mol. Biol. Cell 19, 5226–5237 (2008).

Mahrhold, S., Rummel, A., Bigalke, H., Davletov, B. & Binz, T. The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves. FEBS Lett. 580, 2011–2014 (2006). References 61, 62 and 63 report that the synaptic vesicle protein SV2 functions as a protein receptor for BoNT/A1 and BoNT/E1.

Mahrhold, S. et al. Identification of the SV2 protein receptor-binding site of botulinum neurotoxin type E. Biochem. J. 453, 37–47 (2013).

Benoit, R. M. et al. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A. Nature 505, 108–111 (2014).

Schiavo, G. Structural biology: dangerous liaisons on neurons. Nature 444, 1019–1020 (2006).

Colasante, C. et al. Botulinum neurotoxin type A is internalized and translocated from small synaptic vesicles at the neuromuscular junction. Mol. Neurobiol. 48, 120–127 (2013).

Harper, C. B. et al. Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J. Biol. Chem. 286, 35966–35976 (2011).

Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006). This paper provides a landmark analysis of the fine structure and molecular composition of synaptic vesicles.

Saheki, Y. & De Camilli, P. Synaptic vesicle endocytosis. Cold Spring Harb. Perspect. Biol. 4, a005645 (2012).

Wohlfarth, K., Goschel, H., Frevert, J., Dengler, R. & Bigalke, H. Botulinum A toxins: units versus units. Naunyn. Schmiedebergs. Arch. Pharmacol. 355, 335–340 (1997).

Rasetti-Escargueil, C., Liu, Y., Rigsby, P., Jones, R. G. & Sesardic, D. Phrenic nerve hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies. Toxicon 57, 1008–1016 (2011).

Sun, S., Tepp, W. H., Johnson, E. A. & Chapman, E. R. Botulinum neurotoxins B and E translocate at different rates and exhibit divergent responses to GT1b and low pH. Biochemistry 51, 5655–5662 (2012).

Ahnert-Hilger, G., Holtje, M., Pahner, I., Winter, S. & Brunk, I. Regulation of vesicular neurotransmitter transporters. Rev. Physiol. Biochem. Pharmacol. 150, 140–160 (2003).

Simpson, L. L., Coffield, J. A. & Bakry, N. Inhibition of vacuolar adenosine triphosphatase antagonizes the effects of clostridial neurotoxins but not phospholipase A2 neurotoxins. J. Pharmacol. Exp. Ther. 269, 256–262 (1994).

Williamson, L. C. & Neale, E. A. Bafilomycin A1 inhibits the action of tetanus toxin in spinal cord neurons in cell culture. J. Neurochem. 63, 2342–2345 (1994). References 75 and 76 show that the acidification of an intracellular compartment by the vesicular ATPase proton pump is a necessary step in nerve intoxication by clostridial neurotoxins.

Sun, S. et al. Receptor binding enables botulinum neurotoxin B to sense low pH for translocation channel assembly. Cell Host Microbe 10, 237–247 (2011).

Montal, M. Botulinum neurotoxin: a marvel of protein design. Annu. Rev. Biochem. 79, 591–617 (2010).

Fischer, A. Synchronized chaperone function of botulinum neurotoxin domains mediates light chain translocation into neurons. Curr. Top. Microbiol. Immunol. 364, 115–137 (2013).

Hoch, D. H. et al. Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes. Proc. Natl Acad. Sci. USA 82, 1692–1696 (1985). This is the first study to describe the formation of ion channels by clostridial neurotoxins in planar lipid bilayers.

Donovan, J. J. & Middlebrook, J. L. Ion-conducting channels produced by botulinum toxin in planar lipid membranes. Biochemistry 25, 2872–2876 (1986).

Blaustein, R. O., Germann, W. J., Finkelstein, A. & DasGupta, B. R. The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers. FEBS Lett. 226, 115–120 (1987).

Koriazova, L. K. & Montal, M. Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nature Struct. Biol. 10, 13–18 (2003).

Fischer, A. & Montal, M. Crucial role of the disulfide bridge between botulinum neurotoxin light and heavy chains in protease translocation across membranes. J. Biol. Chem. 282, 29604–29611 (2007). This study shows that the disulphide bond that connects the L chain and H chain of BoNT/A1 and BoNT/E1 must be reduced on the cytosolic side of the synaptic vesicle to release the L chain metalloprotease into the cytosol.

Fischer, A. & Montal, M. Single molecule detection of intermediates during botulinum neurotoxin translocation across membranes. Proc. Natl Acad. Sci. USA 104, 10447–10452 (2007).

Sheridan, R. E. Gating and permeability of ion channels produced by botulinum toxin types A and E in PC12 cell membranes. Toxicon 36, 703–717 (1998).

Dalla Serra, M. et al. Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv. syringae, in planar lipid membranes. Mol. Plant. Microbe Interact. 12, 401–409 (1999).

Fischer, A. et al. Molecular architecture of botulinum neurotoxin E revealed by single particle electron microscopy. J. Biol. Chem. 283, 3997–4003 (2008).

Bade, S. et al. Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J. Neurochem. 91, 1461–1472 (2004).

Galloux, M. et al. Membrane Interaction of botulinum neurotoxin A translocation (T) domain. The belt region is a regulatory loop for membrane interaction. J. Biol. Chem. 283, 27668–27676 (2008).

Fischer, A. et al. Bimodal modulation of the botulinum neurotoxin protein-conducting channel. Proc. Natl Acad. Sci. USA 106, 1330–1335 (2009).

Pirazzini, M. et al. Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain. FEBS Lett. 587, 3831–3836 (2013).

Schiavo, G. et al. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, 832–835 (1992). This study shows that VAMP has an essential role in neurotransmitter release and that both tetanus toxin and BoNT/B cleave the same protein at the same site, despite the different clinical symptoms that they cause.

Schiavo, G., Papini, E., Genna, G. & Montecucco, C. An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin. Infect. Immun. 58, 4136–4141 (1990).

de Paiva, A. et al. A role for the interchain disulfide or its participating thiols in the internalization of botulinum neurotoxin A revealed by a toxin derivative that binds to ecto-acceptors and inhibits transmitter release intracellularly. J. Biol. Chem. 268, 20838–20844 (1993).

Eswaramoorthy, S., Kumaran, D., Keller, J. & Swaminathan, S. Role of metals in the biological activity of Clostridium botulinum neurotoxins. Biochemistry 43, 2209–2216 (2004).

Fu, F. N., Busath, D. D. & Singh, B. R. Spectroscopic analysis of low pH and lipid-induced structural changes in type A botulinum neurotoxin relevant to membrane channel formation and translocation. Biophys. Chem. 99, 17–29 (2002).

Puhar, A., Johnson, E. A., Rossetto, O. & Montecucco, C. Comparison of the pH-induced conformational change of different clostridial neurotoxins. Biochem. Biophys. Res. Commun. 319, 66–67 (2004).

Pirazzini, M. et al. Time course and temperature dependence of the membrane translocation of tetanus and botulinum neurotoxins C and D in neurons. Biochem. Biophys. Res. Commun. 430, 38–42 (2013).

Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

Sankaranarayanan, S. & Ryan, T. A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nature Cell Biol. 2, 197–204 (2000).

Eisenberg, M., Gresalfi, T., Riccio, T. & McLaughlin, S. Adsorption of monovalent cations to bilayer membranes containing negative phospholipids. Biochemistry 18, 5213–5223 (1979).

Nordera, P., Serra, M. D. & Menestrina, G. The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential. Biophys. J. 73, 1468–1478 (1997).

Deutsch, J. W. & Kelly, R. B. Lipids of synaptic vesicles: relevance to the mechanism of membrane fusion. Biochemistry 20, 378–385 (1981).

Ledeen, R. W., Diebler, M. F., Wu, G., Lu, Z. H. & Varoqui, H. Ganglioside composition of subcellular fractions, including pre- and postsynaptic membranes, from Torpedo electric organ. Neurochem. Res. 18, 1151–1155 (1993).

Bychkova, V. E., Pain, R. H. & Ptitsyn, O. B. The 'molten globule' state is involved in the translocation of proteins across membranes. FEBS Lett. 238, 231–234 (1988).

Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E. & Razgulyaev, O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262, 20–24 (1990).

van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H. & Pattus, F. A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A. Nature 354, 408–410 (1991). This paper provides the first evidence that a bacterial toxin adopts a molten globular state during membrane translocation.

Kukreja, R. & Singh, B. Biologically active novel conformational state of botulinum, the most poisonous poison. J. Biol. Chem. 280, 39346–39352 (2005).

Meyer, Y., Buchanan, B. B., Vignols, F. & Reichheld, J. P. Thioredoxins and glutaredoxins: unifying elements in redox biology. Annu. Rev. Genet. 43, 335–367 (2009).

Hanschmann, E. M., Godoy, J. R., Berndt, C., Hudemann, C. & Lillig, C. H. Thioredoxins, glutaredoxins, and peroxiredoxins — molecular mechanisms and health significance: from cofactors to antioxidants to redox signaling. Antioxid. Redox Signal. 19, 1539–1605 (2013).

Berndt, C., Lillig, C. H. & Holmgren, A. Thioredoxins and glutaredoxins as facilitators of protein folding. Biochim. Biophys. Acta 1783, 641–650 (2008).

Pirazzini, M. et al. The thioredoxin reductase–thioredoxin system is involved in the entry of tetanus and botulinum neurotoxins in the cytosol of nerve terminals. FEBS Lett. 587, 150–155 (2013). This study provides the first evidence that the thioredoxin reductase–thioredoxin protein disulphide-reducing system reduces the inter-chain disulphide bond of clostridial neurotoxins in the neuronal cytosol.

Dekker, C., Willison, K. R. & Taylor, W. R. On the evolutionary origin of the chaperonins. Proteins 79, 1172–1192 (2011).

Sudhof, T. C. & Rizo, J. Synaptic vesicle exocytosis. Cold Spring Harb. Perspect. Biol. 3, a005637 (2011).

Pantano, S. & Montecucco, C. The blockade of the neurotransmitter release apparatus by botulinum neurotoxins. Cell. Mol. Life Sci. 71, 793–811 (2014).

Binz, T. Clostridial neurotoxin light chains: devices for SNARE cleavage mediated blockade of neurotransmission. Curr. Top. Microbiol. Immunol. 364, 139–157 (2013).

Hayashi, T. et al. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13, 5051–5061 (1994). This study shows that VAMP, SNAP25 and syntaxin form a tight coiled-coil complex that is resistant to proteolysis by tetanus and botulinum neurotoxins and to SDS.

Sutton, R. B., Fasshauer, D., Jahn, R. & Brunger, A. T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347–353 (1998). This fundamental paper describes the atomic coiled-coil structure of the SNARE complex and its importance for neurotransmitter release.

Megighian, A. et al. Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction. J. Cell Sci. 126, 3134–3140 (2013).

Kalb, S. R. et al. Discovery of a novel enzymatic cleavage site for botulinum neurotoxin F5. FEBS Lett. 586, 109–115 (2012).

Schiavo, G., Shone, C. C., Rossetto, O., Alexander, F. C. & Montecucco, C. Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin. J. Biol. Chem. 268, 11516–11519 (1993).

Whitemarsh, R. C. et al. Characterization of botulinum neurotoxin a subtypes1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro. Infect. Immun. 81, 3894–3902 (2013).

Wang, D. et al. Comparison of the catalytic properties of the botulinum neurotoxin subtypes A1 and A5. Biochim. Biophys. Acta 1834, 2722–2728 (2013).

Shoemaker, C. B. & Oyler, G. A. Persistence of botulinum neurotoxin inactivation of nerve function. Curr. Top. Microbiol. Immunol. 364, 179–196 (2013).

Whitemarsh, R. C., Tepp, W. H., Johnson, E. A. & Pellett, S. Persistence of botulinum neurotoxin A subtypes 1–5 in primary rat spinal cord cells. PLoS ONE. 9, e90252 (2014).

Naumann, M. et al. Evidence-based review and assessment of botulinum neurotoxin for the treatment of secretory disorders. Toxicon 67, 141–152 (2013).

Wang, J. et al. A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic. J. Biol. Chem. 286, 6375–6385 (2011).

Guo, J., Pan, X., Zhao, Y. & Chen, S. Engineering clostridia neurotoxins with elevated catalytic activity. Toxicon 74c, 158–166 (2013).

Ma, L. et al. Single application of A2 NTX, a botulinum toxin A2 subunit, prevents chronic pain over long periods in both diabetic and spinal cord injury-induced neuropathic pain models. J. Pharmacol. Sci. 119, 282–286 (2012).

Chen, S. & Barbieri, J. T. Engineering botulinum neurotoxin to extend therapeutic intervention. Proc. Natl Acad. Sci. USA 106, 9180–9184 (2009).

Wang, D. et al. Syntaxin requirement for Ca 2+ -triggered exocytosis in neurons and endocrine cells demonstrated with an engineered neurotoxin. Biochemistry 50, 2711–2713 (2011).

Franciosa, G., Ferreira, J. L. & Hatheway, C. L. Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. J. Clin. Microbiol. 32, 1911–1917 (1994).

Luquez, C., Raphael, B. H. & Maslanka, S. E. Neurotoxin gene clusters in Clostridium botulinum type Ab strains. Appl. Environ. Microbiol. 75, 6094–6101 (2009).

Carter, A. T., Stringer, S. C., Webb, M. D. & Peck, M. W. The type F6 neurotoxin gene cluster locus of group II Clostridium botulinum has evolved by successive disruption of two different ancestral precursors. Genome Biol. Evol. 5, 1032–1037 (2013).

Dover, N. et al. Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: BoNT/A2, BoNT/F4 and BoNT/F5. PLoS ONE 8, e61205 (2013).

Jahn, R. & Fasshauer, D. Molecular machines governing exocytosis of synaptic vesicles. Nature 490, 201–207 (2012).

Harlow, M. L. et al. Alignment of synaptic vesicle macromolecules with the macromolecules in active zone material that direct vesicle docking. PLoS ONE 8, e69410 (2013).

Zhai, R. G. & Bellen, H. J. The architecture of the active zone in the presynaptic nerve terminal. Physiol. (Bethesda) 19, 262–270 (2004).

Kasai, H., Takahashi, N. & Tokumaru, H. Distinct initial SNARE configurations underlying the diversity of exocytosis. Physiol. Rev. 92, 1915–1964 (2012).

Chernomordik, L. V. & Kozlov, M. M. Mechanics of membrane fusion. Nature Struct. Mol. Biol. 15, 675–683 (2008).

Middlebrook, J. L. & Brown, J. E. Immunodiagnosis and immunotherapy of tetanus and botulinum neurotoxins. Curr. Top. Microbiol. Immunol. 195, 89–122 (1995).

Fairweather, N. F., Lyness, V. A. & Maskell, D. J. Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli. Infect. Immun. 55, 2541–2545 (1987).

Byrne, M. P. & Smith, L. A. Development of vaccines for prevention of botulism. Biochimie 82, 955–966 (2000).

Smith, L. A. Botulism and vaccines for its prevention. Vaccine 27, D33–D39 (2009).

Karalewitz, A. P.-A. & Barbieri, J. T. Vaccines against botulism. Curr. Opin. Microbiol. 15, 317–324 (2012).

Arnon, S. S., Schechter, R., Maslanka, S. E., Jewell, N. P. & Hatheway, C. L. Human botulism immune globulin for the treatment of infant botulism. N. Engl. J. Med. 354, 462–471 (2006).

Garcia-Rodriguez, C. et al. Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin. Nature Biotech. 25, 107–116 (2007).

Lou, J. et al. Affinity maturation of human botulinum neurotoxin antibodies by light chain shuffling via yeast mating. Protein Eng. Des. Sel. 23, 311–319 (2010).

Cheng, L. W., Stanker, L. H., Henderson, T. D., Lou, J. & Marks, J. D. Antibody protection against botulinum neurotoxin intoxication in mice. Infect. Immun. 77, 4305–4313 (2009).

Conway, J. O., Sherwood, L. J., Collazo, M. T., Garza, J. A. & Hayhurst, A. Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents. PLoS ONE 5, e8818 (2010).

Thanongsaksrikul, J. & Chaicumpa, W. Botulinum neurotoxins and botulism: a novel therapeutic approach. Toxins (Basel) 3, 469–488 (2011).

Li, B. et al. Small molecule inhibitors as countermeasures for botulinum neurotoxin intoxication. Molecules 16, 202–220 (2011).

Lee, K. et al. Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex R. Science 344, 1405–1410 (2014).


Mining the botulinum genome

The toxin that causes botulism is the most potent that we know of. Eating an amount of toxin just 1000 th the weight of a grain of salt can be fatal, which is why so much effort has been put into keeping Clostridium botulinum, which produces the toxin, out of our food.

The Institute of Food Research on the Norwich Research Park has been part of that effort through studying the bacteria and the way they survive, multiply and cause such harm. In new research, IFR scientists have been mining the genome of C. botulinum to uncover new information about the toxin genes.

There are seven distinct, but similar, types of botulinum neurotoxin, produced by different strains of C. botulinum bacteria. Different sub-types of the neurotoxin appear to be associated with different strains of the bacteria. Genetic analysis of these genes will give us information about how they evolved.

Dr Andy Carter, working in Professor Mike Peck's research group, used data generated from sequencing efforts at The Genome Analysis Centre, on the Norwich Research Park. Andy compared the genome sequence of five different C. botulinum strains, all from the same group and all producing the same sub-type of neurotoxin.

An initial finding was that the five strains were remarkably similar in the area of the genome containing the neurotoxin gene. This suggests that the bacteria picked up the gene cluster in a single event, sometime in the past. Bacteria commonly acquire genes, or gene clusters, from other bacteria through this horizontal gene transfer. It is a way that bacteria have evolved to share 'weapons', such as antibiotic activity or the ability to produce toxins. To find out more about how C. botulinum acquired its own deadly weapon, Andy delved deeper into the genome sequence.

Like fossils of long lost organisms, Andy found, in the same region of the genome, evidence of two other genes for producing two of the other types of neurotoxin. Although these gene fragments are completely non-functional, finding them in the same place in the genome as the functional neurotoxin gene cluster is significant as it suggests that this region of the genome could be a 'hotspot' for gene transfer.

Looking to either side of the neurotoxin gene cluster uncovered more evidence supporting the hotspot idea. When the gene cluster inserted into the C. botulinum genome, it cut in two another gene. This gene is essential for the bacteria to replicate its DNA, so why does destroying it not prove fatal? C. botulinum was unaffected by this because contained in the segment of imported DNA was another version of the chopped-up gene.

Perhaps this is pointing us to the way C. botulinum first picks up its lethal weapon. This should help us prepare against the emergence of new strains, and may even one day help us disarm this deadly foe.

The research was funded by the Biotechnology and Biological Sciences Research Council and published in the journal Genome Biology and Evolution Advance.


Pathogenesis

Transmission:

It is ubiquitous in nature, widely distributed as a saprophyte in soil, animal manure, vegetables, and sea mud. Homemade canned foods, condiments, and fish products are the most common sources of infection with C. botulinum. Ingestion of contaminated honey is the major cause of infant botulism.

Insufficient cooking temperature followed by packaging in anaerobic conditions facilitates the germination of spores and synthesis neurotoxins.

Mechanism of action of Botulinum toxin (BoNT)

Clostridium botulinum is non-invasive. Its pathogenesis is due to the production of powerful neurotoxin ‘botulinum toxin’ (BoNT), probably the most toxic substance known to be lethal to mankind. It produces flaccid paralysis. There are 7 serological types of botulinum neurotoxin labeled as types A, B, C [C1 C2], D, E, F, and G. Human botulism is caused mainly by types A, B, E and F (rarely).

C. botulinum toxin is categorized as a potential bioterrorism agent but botox is in use to smooth facial wrinkles.

After entry (either ingested, inhaled, or produced in a wound), botulinum toxin is transported via the blood to peripheral cholinergic nerve terminals. The most common nerve terminal sites are neuromuscular junctions, postganglionic parasympathetic nerve endings, and peripheral ganglia. It does not affect the CNS.

In normal condition: Upon stimulation of peripheral and cranial nerves, acetylcholine is normally released from vesicles at the neural side of the motor endplate. Acetylcholine then binds to specific receptors on the muscle, inducing contraction.

Mechanism of Botulinum toxin
(Image source: lumenlearning.com)

Botulinum toxin acts by binding to synaptic vesicles of cholinergic nerves, thereby preventing the release of acetylcholine (Ach) at the peripheral nerve endings, including neuromuscular junctions. This results in a lack of stimulus to the muscle fibers, irreversible relaxation of the muscles, and flaccid paralysis.

As botulinum toxin produces flaccid paralysis it can be used therapeutically for the treatment of spasmodic conditions such as strabismus (misaligned eyes), blepharospasm (uncontrollable blinking), and myoclonus.

Clinical manifestations

  1. Diplopia (double vision) or blurring of vision
  2. Dysphagia (difficulty swallowing)
  3. Dysarthria (difficulty in speech) or slurring of speech
  4. Descending symmetric flaccid paralysis of voluntary muscles.
  5. Decreased deep tendon reflexes
  6. Fatigue
  7. Dizziness
  8. Nausea
  9. Constipation
  10. Respiratory muscle paralysis may lead to death.

There is no sensory or cognitive deficits

Types of Botulism

  1. Foodborne botulism: It results from the consumption of foods contaminated with preformed botulinum toxin such as homemade canned food.
  2. Wound botulism: It is a systemic intoxication resulting from the growth of C. botulinum and toxin production in the wounds. It presents like foodborne botulism except for the absence of gastrointestinal features.
  3. Infant botulism: Infant botulism is much milder than the adult version. It results from the ingestion of food (usually honey) contaminated with spores of C. botulinum by children ≤1 year of age. Spores germinate in the intestine, and the vegetative cells secrete botulinum toxin. Clinical manifestations include the inability to suck and swallow, weakened voice, ptosis, floppy neck, and extreme weakness hence called floppy child syndrome. It is a self-limiting disease prognosis is excellent if managed by supportive care and assisted feeding.

Spores do not normally germinate in adult intestine, however may germinate in the intestine of infants.


5 POSTULATE IV

“Molecular potency” is difficult to objectively quantify for commercially available toxins.

Methods of comparison of molecular potency for commercially available toxins include comparing independent trial data comparing different toxins in different subjects in a single trial (noninferiority) bilateral comparisons of different toxins in a single subject and direct measurement of toxin pg and activity. There have been a number of trials attempting to compare molecular potency among toxins however, the data make it difficult to form absolute conclusions. The main reason for this is that the units of toxin products are proprietary measurements and dependent on the type of assay used. This makes every toxin unique and impossible to directly compare with each other. In addition, the LD50 is manufacturer-dependent and based on mouse models rather than human ones and the amount of 150 kDa neurotoxin, availability, and activity vary from product to product. 14 This further complicates direct methods of comparing potency among products. Keeping this in mind, the closest we can get to comparing potencies is by evaluating each toxin's clinical effect based on the FDA-approved units, which are still confounded by differences in test subjects despite rigorous inclusion and exclusion criteria and rating scales that may not translate into actual clinical application. Each study also has its own unique endpoints. This makes comparing the efficacy of toxin brands incredibly difficult. The majority of BoNT-A comparison studies have been focused on AbobotulinumtoxinA vs OnabotulinumtoxinA and differ in their reports of efficacy, time of onset, and duration between the two.

Some of the most often quoted comparisons of commercial toxins are onset, duration, and adverse events obtained in separate FDA approval trials. 56, 108, 109 However, since different FDA trials use different protocols and efficacy scales and are performed by different investigators, it is impossible to use them as accurate comparators of molecular potencies. According to the FDA Guidance to Industry, assessment scales should also be ordinal, static, reproducible, and include only a limited number of distinct and clinically meaningful categories, preferably with a photonumeric guide for patients and investigators. 110 Common assessment tools such as the facial wrinkle scale and GL severity score are 4-point photonumeric ordinal scales that ranging from no wrinkling to severe wrinkling and have shown good inter- and intraobserver reproducibility. 111, 112 The 5-point photonumeric scale developed by Caruthers and Carruthers is a good example. By including a midpoint, it allows for grading of a continuous process such as aging. 113-115 Most FDA studies, however, use their own proprietary FDA-approved, validated scales that differ from manufacturer to manufacturer.

“Side-by-side” methods of comparing neurotoxins are much more accurate and have suggested differences in potency between BoNT-A products. In this scenario, one group of patients receives one particular toxin while another group receives another. In a 150-day, multicenter, double-blind, single-dose (corresponding to FDA-approved doses) noninferiority trial comparing PrabotulinumtoxinA to OnabotulinumtoxinA at the same 20U dose (approved dose for GL) and placebo, a 5:5:1 ratio of 540 patients were administered 0.1mL of the corresponding treatment to each of the 5 glabellar injection points. Although not quite reaching statistical significance, there was indication of increased duration of effect for PrabotulinumtoxinA. 71

Other side-by-side trials have compared potency of OnabotulinumtoxinA and AbobotulinumtoxinA. The majority of these trials are weak, present conflicting conclusions regarding potency, and often compare nonequivalent doses of drug. One study compared the FDA-approved doses of 20 U of OnabotulinumtoxinA with 50 U of AbobotulinumtoxinA (1:2.5 dose ratio) and compared glabellar line severity at 12 and 16-week endpoints. Results showed a 1 point or greater grade improvement in 77% and 53% of patients for weeks 12 and 16 respectively among OnabotulinumtoxinA-treated patients and 59% and 28% improvement in AbobotulinumtoxinA-treated patients. 68 The study, however, enrolled only a small number of patients and included mostly younger patients that may require higher doses of drug due to stronger corrugators compared to older patients. 68

Split-face studies seem to provide the most direct and accurate method for clinically comparing toxin potency because they allow for patients to act as their own control, using reproducible, identical techniques and objective measurements. Recent studies have compared the effect of different BoNT-A products on frontalis muscle in a split-face design. In a randomized, double-blind trial of 20 female subjects, 5 units of AbobotulinumtoxinA and 2 units of OnabotulinumtoxinA (reconstituted in identical 2.4 mL volumes) were injected on contralateral sides of each frontalis muscle. Results showed OnabotulinumtoxinA to have a median time to onset of effect of 3.8 days and AbobotulinumtoxinA to have a median time of onset of 1.8 days. OnabotulinumtoxinA also displayed a median duration of effect of 84 days while AbobotulinumtoxinA had a median duration of 104 days. 44, 55, 116 This trial is a good example of an attempt to quantify molecular potency through clinical measurement, and the differences in onset and duration are likely due to having larger quantities of active 150kDa neurotoxin molecules in 50 units of AbobotulinumtoxinA compared with 20 units of OnabotulinumtoxinA. Additionally, split muscle studies such as this one are free of subject to subject differences in facial anatomy.

The frontalis model utilized by Nestor and Ablon has been demonstrated as an effective method in comparing differences in time to onset between BoNT-A formulations. 44 While many patients report toxin effect as early as the first day of injection, prior studies often do not capture this data until at least 1 week or more postinjection. Nestor and Ablon incorporated a novel, more sensitive and objective assessment that captured the onset of effect as early as 6 hours post–BoNT-A injection. They utilized a Frontalis Activity Measurement Standard (FMS) and 4-point Frontalis Rating Scale (FRS) to compare the onset of effect of AbobotulinumtoxinA to OnabotulinumtoxinA injected into contralateral sides of the frontalis muscle of the same patient. Among 20 subjects, the study demonstrated that time to onset in fact is not equivalent among the different brands of BoNT-A. Using a dose-unit ratio of 2.5:1 with identical injection volumes, onset of effect was measurable within the first 18 hours in 90% of frontalis sides treated with AbobotulinumtoxinA but only 20% of sides treated with OnabotulinumtoxinA. At all time points, AbobotulinumtoxinA demonstrated significantly earlier onset than OnabotulinumtoxinA, as shown in Figure 2. 44, 116

The FMS has been an effective scale for comparing different toxin products in split-face studies. 44, 116 It allows for direct bilateral comparison of different products, dosing, and technique on a single patient through objective quantification of changes in muscle activity because it requires investigators to measure differences in frontalis height at rest and maximum elevation. 44, 116 The other advantage is that it allows for measurement of field of effect without having to use the Minor's test, a conventional assessment technique that compares degree of anhidrosis among products. 44, 116, 117 The FMS assessment includes a series of photographs using the same camera settings and lighting conditions with a rest period of 1 minute between photographs. The onset of action using this scale has been detected as early as 6 hours after injection. The FMS was utilized in a split-face comparison of AbobotulinumtoxinA vs OnabotulinumtoxinA and allowed for precise and accurate comparison of 2 different BoNT-A products not previously reported in the literature, as seen in Figure 3. 55 Others confirm this observation of the difference in molecular potency of AbobotulinumtoxinA compared to OnabotulinumtoxinA in regard to time of onset. One study, using dose ratios of 2.5:1 and 3:1 (AbobotulinumtoxinA:OnabotulinumtoxinA) and a generally higher dose of AbobotulinumtoxinA than OnabotulinumtoxinA, found a mean difference in glabellar lines (GL) of 0.52 days (P < .0001). 89 In this study, patients treated with AbobotulinumtoxinA reported noticeable differences in glabellar lines on Day 1 more frequently than patients treated with OnabotulinumtoxinA (28% vs 17%, respectively). The onset of effect on lateral canthal lines (LCL) was also shorter among AbobotulinumtoxinA-treated patients compared to OnabotulinumtoxinA by a mean of 0.33 days (P = .0025). Duration of effect on GL and LCL was also shown to be superior among patients treated with AbobotulinumtoxinA rather than OnabotulinumtoxinA, with a larger proportion of patients retaining a response by 4 and 5 months. These results accounted for higher satisfaction rates among patients treated with AbobotulinumtoxinA vs OnabotulinumtoxinA.

Finally, a direct molecular method of comparison is another way in which discrepancies between toxin potencies among manufacturers have been highlighted. One study compared the quantity and light chain (LC) activity of BoNT-A in three commercial BoNT-A products (Dysport Botox Xeomin). Direct measurements of the quantity of the mean active 150 kDa BoNT-A content for the FDA-approved glabella dosing have found that IncobotulinumtoxinA, OnabotulinumtoxinA, and AbobotulinumtoxinA have 80.6 pg/20 XU, 180.8 pg/20 BU and 301.1 pg/50 DU, respectively. These were measured with ELISA and activity measured by EndoPep assays which demonstrated equivalent light chain activity per nanogram of neurotoxin among all three products. Differences in treatment duration of action may, therefore, be due to differences in the actual quantity of neurotoxin molecules injected rather than the LD50 determined potency of the toxin. 57

5.1 Subjective scales

While objective scales have aided in determining efficacy, subjective scales such as the subjective global assessment and FACE-Q validated, patient-reported outcome questionnaire have been useful in assessing patient satisfaction as well. 111, 118 These scales have been useful in identifying an improvement in patient-reported outcomes as dosing recommendations have changed. 51 In the past, the aim of BoNT-A administration was to achieve total muscle immobilization. This, however, compromised facial expressiveness as seen in subjective scales. Since then, ideal dosing has decreased to provide patients with a more natural and balanced look while still diminishing unwanted lines. 119 BoNT-A treatment has also been associated with improvement in depression in depressed patients. 120-122 Subjective evaluations have therefore become increasingly important to achieve because patient satisfaction influences treatment choice but do not give an accurate representation of molecular potency.

5.2 Comparing duration of effect

The duration of effect is probably the most important metric of molecular potency although it comes with the caveat that increased duration is directly associated with a more frozen appearance. The frontalis model was again used in a second study which utilized both the FMS and an additional standard Frontalis Rating Scale (FRS) which quantified degree of clinical effect or level of “frozen” appearance on a scale of partial, full, to complete efficacy. Still, the FMS proved to be more sensitive in measuring effect and was able to detect changes in appearance earlier than the FRS. In correlation with the prior study, AbobotulinumtoxinA appeared to show greater molecular potency at FDA-approved toxin ratios (50 units of AbobotulinumtoxinA and 20 units of OnabotulinumtoxinA) to OnabotulinumtoxinA in terms of maintaining duration of all degrees of efficacy among a higher proportion of frontalis sides (Table 2). 116

Measurement Efficacy AbobotulinumtoxinA OnabotulinumtoxinA Significance
FRS Partial 160 145 Not significant
Full 119 77 P = .003
Complete 63 44 P = .01
FMS Partial 105 99 P = .006
Full 103 87 P = .003
Complete 72 56 P = .01

Study Helps Explain Why Botulinum Toxin Is So Deadly

A pilot without a map can locate an airport by first finding a nearby landmark, like a big river, and then searching for the airport.

New research from the University of Wisconsin School of Medicine and Public Health (SMPH) and Scripps Research Institute shows how the astonishingly powerful botulinum toxin uses a similar strategy to latch onto nerve cells, the first step in inactivating them.

The research helps explain how the toxin first attaches to a receptor on the surface of a nerve cell, then looks for a second type of receptor that is nearby. Once the toxin links to this second receptor, it can enter the nerve cell and break a protein needed to deliver molecules that can signal other nerve cells.

By blocking this signaling molecule, tiny amounts of botulinum toxin can cause paralysis and even death through respiratory failure. The bacteria that makes this toxin grows in soil, and can be found inside cans of food that were improperly processed. Botulinum toxin is the reason for the extreme danger from bulging cans of food.

Researchers have been working on the unique nerve-blocking ability of the seven individual botulinum toxins for decades, says botulinum expert Edwin Chapman, UW-Madison professor of physiology and a Howard Hughes Medical Institute investigator. "A major question is how the toxin enters neurons," he says.

The research was a close collaboration with Ray Stevens of Scripps, who crystallized the structure that forms when botulinum toxin links to the protein receptor on a nerve cell.

"This is the first paper to show in atomic detail the structure of botulinum neurotoxin touching the receptor on the surface of the neuron," Chapman says. "The toxin has to bind to the neuron it wants to poison. This is a snapshot of the first stage of that poisoning."

The report on the work, in the journal Nature this week, identified a short section on the protein receptor as the exact spot where botulinum toxin grips the cell immediately before entering it.

UW-Madison has long been a center of botulism research. In 2003, Min Dong, a post-doctoral fellow in Chapman's lab, showed that a known protein receptor for one botulinum toxin was a key point of entry into the nerve cell. Dong shares first authorship on the current study along with Qing Chai and Joseph Arndt of Scripps.

The Nature paper is an elaboration on that 2003 discovery, which was published in The Journal of Cell Biology. Stevens's lab bombarded a crystal of the toxin bound to a small sub-region of the primary receptor with X-rays, then measured the reflections to portray the toxin and the receptor bound in deadly embrace.

The research could have several practical applications. Botulinum toxin is a potential biological weapon, so the U.S. military is interested in finding anti-toxins to protect soldiers -- molecules that attach to the binding site on the toxin or on the cell. The search for such a blocking molecule becomes easier now that the exact structure of the link between the toxin and the nerve cell are known.

Better knowledge of botulinum toxin's structure could also enhance the growing number of treatments that use the toxin to block nerve signals. The medical treatments "are not just for wrinkles," Chapman says. "People with paralysis get spasms in the muscles that are shut off, and this could solve that. In a wide variety of dystonias, where spasms can cause really severe pain, this can relax the muscles."

A third potential benefit is further down the line. After the researchers found the binding site on the protein receptor, they varied it until the toxin could no longer bind to it. If a mutated toxin was made to attach to the mutated receptor, the combination might target botulinum toxin against over-active cells in the body, Chapman suggests.

Using genetic engineering, "you might be able to sensitize whatever cell you want to the toxin," he says. Theoretically, such a treatment could be used to slow mucus production in the lungs of cystic fibrosis patients, or to attack hyperactive cells in a wide range of other disorders.

Overall, the research improves our knowledge of a devilishly clever toxin, says Dong. Botulinum is an enzyme - a biological catalyst -- that can move through a cell, breaking one protein molecule and quickly attacking another. Botulinum toxin attacks communications between nerve cells, "one of the most sensitive parts of the animal physiology," Dong says. "That provides an efficient way to immobilize an animal, far easier than targeting muscles directly."

Another reason for botulinum toxin's extraordinary power becomes clear from this study, Dong says. The toxin is only able to attach to a nerve cell that is working. "If the synapse between two nerve cells is not active, all the receptors will be hiding inside the cells. But a synapse that controls a very important muscle must be firing all the time, and it will be exposing more receptors, and the toxin will therefore target them."

Botulinum toxin, he says, "Only goes where it can be effective. It's like a smart bomb."


About Botulism

This illustration depicts a three-dimensional (3D) computer-generated image of a group of anaerobic, spore-forming, Clostridium sp. organisms.

Botulism (&ldquoBOT-choo-liz-um&rdquo) is a rare but serious illness caused by a toxin that attacks the body&rsquos nerves and causes difficulty breathing, muscle paralysis, and even death. This toxin is made by Clostridium botulinum and sometimes Clostridium butyricum and Clostridium baratii bacteria. These bacteria can produce the toxin in food, wounds, and the intestines of infants.

The bacteria that make botulinum toxin are found naturally in many places, but it&rsquos rare for them to make people sick. These bacteria make spores, which act like protective coatings. Spores help the bacteria survive in the environment, even in extreme conditions. The spores usually do not cause people to become sick, even when they&rsquore eaten. But under certain conditions, these spores can grow and make one of the most lethal toxins known. The conditions in which the spores can grow and make toxin are:

  • Low-oxygen or no oxygen (anaerobic) environment
  • Low acid
  • Low sugar
  • Low salt
  • A certain temperature range
  • A certain amount of water

For example, improperly home-canned, preserved, or fermented foods can provide the right conditions for spores to grow and make botulinum toxin. When people eat these foods, they can become seriously ill, or even die, if they don&rsquot get proper medical treatment quickly.


Watch the video: Can botulinum toxin benefit rosacea patients? (January 2022).