Botulinum toxin (BoNT) types, subtypes, and their main properties including enzymatic substrates and cleavage sites, as well as the neurotoxin-producing microorganisms with their main physiological properties and involvement in naturally acquired botulism.
Botulinum toxins (BoNTs) are the most potent toxins and are responsible for botulism, which is a neurological disease in man and animals. Botulism is characterized by flaccid paralysis and inhibition of secretions. BoNTs are produced by distinct clostridial species including Clostridium botulinum that consist in four physiological and genetic groups, atypical strains of C. baratii and C. butyricum. Recently, nonclostridial bacteria have been found to synthesize BoNTs. The particularity of BoNTs is to associate with nontoxic proteins to form large-size complexes that are resistant to acidic pH and protease degradation of the digestive tract. BoNTs are divided into 10 types based on neutralization by specific antisera and into more than 40 subtypes according to their sequence variations. All BoNTs retain a common core structure and mode of action, which consists in the inhibition of neurotransmitter release, notably acetylcholine. Human botulism occurs in three main forms: foodborne botulism, botulism by intestinal colonization including infant botulism, and wound botulism. In France, type B foodborne botulism is the most prevalent form, resulting from the traditional consumption of pork products such as home-made cured ham. Albeit less frequent, human botulism is still present in France including diverse types and origins.
- botulinum toxin
- Clostridium botulinum
- flaccid paralysis
Botulinum toxins (BoNTs) are the most potent toxins among bacterial, animal, and plant toxins. Indeed, the lethal activity as tested in laboratory animals by determining the lethal dose 50% (LD50) is the lowest compared to that of all other toxins. Because of its extreme lethal potency, BoNTs are considered as the greatest threat of toxin weapon and are classified as Category A threat agent by the Centers for Disease Control and Prevention Select Agent Program . In the natural conditions, BoNTs are responsible for a neurological disease in man and animals, botulism, which is characterized by flaccid paralysis and inhibition of secretions. Outbreaks of animal botulism are worldwide distributed and cause important economic losses, notably in cattle and farmed birds. Human botulism is much rarer than animal botulism, but it is a severe disease often fatal without treatment. Human botulism is the most severe food poisoning, and botulism surveillance by health and food authorities is performed in most of the countries in order to rapidly identify and withdraw contaminated foods and also to address recommendations to industrials and consumers regarding hygiene and food preservation practices. However, the paralytic effects of BoNTs are used in the treatment of numerous diseases including muscle hyperactivity such as dystonia, strabismus, limb spasticity, sphincter dysfunction, or hypersecretion (hyperhidrosis, hypersialorrhea, and drooling in neurodegenerative diseases), but also in the treatment of pain and in cosmetology. BoNTs are largely used as therapeutic drugs and are one of the drugs that have the most numerous medical indications [2, 3].
Botulism was described in the second part of the eighteenth century and at the beginning of the nineteenth century by Steinbuch (1817) and Kerner (1817–1822). Both described a particular form of foodborne poisoning due to ingestion of a “sausage poison.” An increased number of fatal food poisoning cases occurred at the end of eighteenth century in the southwest German region of Wurtenberg due to a decline in hygiene of rural food productions subsequently to Napoleonic war perturbations. The paralytic disease was mainly associated to the consumption of blood sausages and was termed “sausage poisoning.” This disease was also known as “Kerner’s disease” and the name “botulism” was coined later in the second half of the nineteenth century from the Latin word
2. Botulinum toxins
BoNTs share a common structure. They are synthesized as a precursor protein (about 150 kDa), which is inactive or weakly active. The precursor that does not contain a signal peptide is released from the bacteria by a yet unknown mechanism. The precursor is proteolytically activated in the extra-bacterial medium either by
The overall sequence identity at the amino acid level between BoNTs ranges from 34 to 97%. Several domains are highly conserved which account for the common mode of action of these toxins including the central domains of L chains containing the catalytic site and the N-terminal half of the H-chains that is involved in the translocation of the L-chain into the cytosol. Thus, a similar mechanism of internalization of the intracellular active domain into target cells is shared by all the clostridial neurotoxins.
2.2. Botulinum complexes
BoNTs associate by non-covalent bounds with non-toxic proteins (ANTPs) produced by
All BoNT complexes contain the non-toxic non-hemagglutinin (NTNH) protein. NTNH is highly conserved. Two main classes of botulinum complexes can be distinguished based on their composition in additional proteins, the botulinum complexes containing hemagglutinins (HAs, including HA33, HA17, and HA70) (HA-BoNT complexes) and those possessing OrfX (including OrfX1, Orfx2, and OrfX3) and P47 proteins (OrfX-BoNT complexes) [17, 20, 21, 22, 23]. The composition and structure of HA-BoNT complexes have been extensively investigated, whereas the OrfX-BoNT complexes are still poorly characterized. The stoichiometry can vary according to the strain, culture conditions (culture media, temperature, period of culture, etc.), and the method of complex preparation .
The 12S or M complex results from the association of a BoNT molecule together with a NTNH at a 1:1 ratio . L HA-BoNT complexes of
The composition and organization of OrfX-BoNT complexes from
NTNHs from the different
BoNT and NTNH share a weak amino acid sequence identity (~20%), but both proteins retain a common structure (Figure 1). NTNH associates with BoNT by non-covalent bonds in a pH-dependent manner to form an interlocked compact M complex, which is resistant to acidic pH and protease degradation, whereas each protein separately is sensitive to proteolysis[23, 25, 26, 37]. Thereby, NTNH is a non-toxic protein which acts as a chaperone protein to protect BoNT. NTNH does not contain the catalytic HExxH motif, but another zinc-binding motif, KCLIK, at the same position. Indeed, NTNH binds one zinc atom per each molecule but exhibits no proteolytic activity . This strongly supports that all NTNH and BoNT variants derive from a common ancestor gene by duplication and subsequent independent reshuffling.
HA33-35 is the most abundant hemagglutinin component of the HA-BoNT complexes. Type A HA35 binds to oligosaccharides containing galactose-β1-4glucose-
More recently, a novel function has been attributed to HA complexes consisting in the disruption of intercellular junctions between intestinal epithelial cells. HAs recognize E-cadherin, which plays a crucial role in basolateral junction. The interaction of HAs with E-cadherin is species and isoform specific. Thereby, HAs directly bind to the extracellular domain of (epithelial) E-cadherin, but not of (neural) N-cadherin, nor (vascular endothelial) VE-cadherin. Type B HAs specifically bind to human, bovine, and mouse E-cadherin but not to that of rat and chicken . This is consistent with the fact that botulism type B is common in humans and is rarely observed in chickens. Type A BoNT complexes also recognize human E-cadherin, whereas type C BoNT complexes do not . The combination of HAs (HA33, HA17, and HA50/70) organized in complex is required for the optimum binding to E-cadherin, whereas individual HAs do not interact with E-cadherin. HAs assemble in a threefold symmetric hetero-dodecameric structure, and the whole HA complex exhibits the highest affinity to E-cadherin. The minimal HA complex interacting with E-cadherin consists of domain 3 of HA70 (Pro-378-Asn-626), one molecule of HA17, and two HA33 molecules . HAs bind to the distal extracellular domain (EC1) of E-cadherin near the cadherin trans-dimer interface . Thus, the HA-binding sites to carbohydrates and E-cadherin are functionally and structurally distinct .
The structures of OrfX2 and P47 are unrelated to that of HAs and show that they belong to the tubular lipid-binding (TULIP) protein superfamily. Thereby, OrfX1 and OrfX2 have been found to bind to phosphatidylinositol . In contrast to HAs, OrfX proteins and OrfX complexes have not been reported to interact with E-cadherin or to alter the intestinal epithelial barrier. This raises the question whether OrfX complexes are involved in BoNT passage through epithelial barriers. In
2.3. Botulinum toxin gene organization
The BoNT and ANTP genes are clustered in close vicinity in a DNA fragment which is called the botulinum locus. BoNT and ANTP genes are organized in two operons. The operon localized in the 3′ part of the botulinum locus contains
The botulinum loci are distributed on different genetic elements, including chromosome, plasmid, or phages depending on the species and strain of Clostridia. In
The location of botulinum locus within chromosome or plasmid seems to occur not at random but at specific sites. Indeed, in strains from group I or II, whose genome sequencing is available, five specific sites of botulinum locus integration have been identified.
Two specific sites of botulinum locus location have been identified on plasmids from group I strains, one contains
The toxin gene location on the various genetic elements chromosome including mobile genetic elements (plasmid, phage) supports horizontal
|Botulinum toxin type||BoNT/A||BoNT/B||BoNT/E||BoNT/F|
|Subtypes||A1, A2, A3, A4, A5, A6, A7, A8||B1, B2, B3, B5, B6, B7, B8||B4||E1, E2, E3, E6, E7, E8, E9, E10, E11, E12||F6||F2, F2, F3, F4, F5, F8|
|Enzymatic substrate (cleavage site)||SNAP25 (QR)||VAMP1, 2, 3 (QF)||SNAP25 (RI)||VAMP1, 2, 3 (QK)|
F5: VAMP2 (LE)
|Main physiological properties||Proteolytic|
Temperature growth: minimum 10–12°C, optimum 30–40°C
Highly heat-resistant spores
Growth at low temperature: minimum 2.5–3°C, optimum 25–30°C
Moderate heat-resistant spores
|Idem group I|
|Botulism||Human, occasionally animal|
|Botulinum toxin type||BuNT/E||BaNT/F||BoNT/C||BoNT/D||BoNT/G||BoNT/H|
|Subtypes||E4, E5||F7||C/D, D/C||G||H or F/A or H/A|
|Enzymatic substrate (cleavage site)||SNAP25 (RI)||VAMP2 (QK)||SNAP25 (RA)|
|VAMP1, 2, 3 (KL)||VAMP1, 2, 3 (AA)||VAMP1, 2, 3 (LE)|
|Main physiological properties||Non-proteolytic|
Temperature growth 37–40°C
|Botulism||Human, animal not reported||Animal, very rare in human||No natural case reported||Human|
|Botulinum toxin type||BoNT/X||BoNT/I or BoNT/Wo||BoNT/J or eBoNT/J or BoNT/En||Cp1 toxin (BoNT homolog)||BoNT/Ba|
|Enzymatic substrate (cleavage site)||VAMP1, 2, 3, 4, 5|
|VAMP2 (WW)||VAMP2 (DL)|
|Bivalent and trivalent|
|Main physiological properties||Group I||Gram-positive bacillus|
|Gram-positive cocci||Gram-negative bacillus|
|Botulism||Infant botulism Japan||No natural botulism case reported||Human botulism|
2.4. Botulinum toxin diversity
BoNTs form a family of diverse proteins which share the common property to induce a flaccid paralysis. Historically, it was found that these toxins can be antigenically distinguished. On the basis of neutralization of the biological effects on small rodents with specific antisera, seven BoNT types (A–G) were identified. Each type-specific antitoxin only neutralizes the corresponding BoNT type. The differences in amino acid sequences range from 37.2 to 69.6% . In 2013, a novel eighth BoNT type called H (or F/A or H/A) has been described from a bivalent
An increased sequencing of
Amino acid sequence variations might impact BoNT biological functions including receptor recognition, the efficiency of entry into cells and persistence, recognition by monoclonal antibodies, and enzymatic activity. For example, BoNT/A2 has been shown to enter more efficiently into neuronal cells than BoNT/A1 and to have a higher affinity for its receptor [77, 78]. BoNT/A2 induces a faster paralysis than BoNT/A1/A4/A5, and BoNT/A3 has a shorter duration of effect . In addition, BoNT/A2 retains a lower immunogenicity . Thus, BoNT/A2 would be a more efficient therapeutic agent than BoNT/A1 [81, 82, 83]. BoNT/A8 binds less efficiently to gangliosides embedded into a membrane and has a lower enzymatic activity than BoNT/A1 . BoNT/B1 binds to synaptotagmin 1 and 2 receptors, whereas BoNT/B2 only recognizes synaptotagmin 2 . In contrast to the BoNT/F subtypes which cleave VAMP1 and VAMP2 at QK site, BoNT/F5 uses a distinct cleavage site (LE)  (Table 1). Monoclonal antibodies against BoNT/B differently recognize the subtypes BoNT/B1 to BoNT/B5 . Similarly, monoclonal antibodies against BoNT/A recognize and/or neutralize the distinct BoNT/A subtypes with variable efficiently [88, 89].
3. Mode of action
BoNTs enter by oral route (foodborne botulism) or are produced directly in the intestine (infant or intestinal botulism) subsequently to a
Each type of BoNT and TeNT recognizes specific receptors on demyelinated terminal nerve endings, mainly through the HCC subdomain. BoNT/A/C/E/F exploit the three isoforms of the vesicle protein SV2 as specific receptors, while BoNT/B and /G bind to synaptotagmin I or II [92, 93, 94, 95, 96, 97, 98]. BoNT/C and BoNT/D interact with gangliosides (GD1b, GT1b) and phosphatidylethanolamine, respectively, by their HCC subdomain . Gangliosides (GD1b, GT1b, and GD2) and SV2A/B/C also mediate the entry of BoNT/D into neurons, but by a different mechanism than that used by BoNT/A and BoNT/E [100, 101]. The role of HCN subdomain, which may interact with phosphatidylinositol phosphates , is still unclear. The co-presence of the
Neurotoxin bound to its receptor is internalized by receptor-mediated endocytosis. Acidification of the vesicle lumen triggers a conformational change of the neurotoxin and subsequent translocation of the L chain into the cytosol. In addition, the disulfide bond between the two chains has a crucial role in the translocation process [103, 104, 105, 106]. Then, the L chain refolds in the neutral pH of the cytosol. Cytosolic translocation factors such as β-COPI are possibly involved in this mechanism, as it has been found for diphtheria toxin [107, 108, 109, 110].
L chains of all clostridial neurotoxins are zinc-metalloproteases that cleave one of the three members of the SNARE proteins. BoNT/B, D, F, and G attack synaptobrevin (or VAMP), BoNT/A and E cleaves SNAP25, and BoNT/C1 cut both SNAP25 and syntaxin. The cleavage sites are different for each neurotoxin. The cleavage of SNARE proteins occurs only when disassembled. Since VAMP, SNAP25, and syntaxin play a major role in the regulated fusion of synaptic vesicles with the plasma membrane at the release sites, their cleavage induces a blockade of the neurotransmitter exocytosis.
SNAP25 cleavage by BoNT/A or BoNT/E alters SNAP25 and synaptotagmin interaction, thus strongly reducing the responsiveness to Ca++ of exocytotic machinery [111, 112, 113, 114]. Indeed, the removal of the nine C-terminal amino acids of SNAP-25 by BoNT/A deeply disrupts the coupling between Ca2+ sensing and the final step in exocytosis . Truncated SNAP-25 can behave as a dominant-negative mutant upon the exocytotic process, suggesting that after BoNT/A treatment, the block of release is due to both functional elimination of SNAP-25 and accumulation of the cleavage product which competitively inhibits exocytosis [115, 116, 117]. In contrast, the blockade of exocytosis by BoNT/E is only due to the elimination of functional SNAP-25 and not to the production of competitive antagonists of SNARE complex formation. Indeed, the inhibition of exocytosis by BoNT/E can be rescued by supplementing the C-terminal portion of SNAP-25 removed by the toxin [118, 119, 120]. Truncation of SNAP-25 by BoNT/E destabilizes the four-helix bundle of the SNARE complex [118, 119], and SNAP-25 truncated by BoNT/E is not retained by syntaxin .
VAMP cleavage abolishes the interaction of VAMP with the adaptor protein AP3 and affects synaptic vesicle recycling
BoNT/C cleaves both syntaxin-1 and SNAP-25, but
Although the physiological properties induced by the cleavage of either VAMP, SNAP25, or syntaxin are not equivalent at the neuromuscular junctions, all the clostridial neurotoxins cause a blockade of the regulated neurotransmission, which varies in intensity and duration according to each neurotoxin type.
4. Epidemiology of botulism in France
4.1. Main clinical forms of human botulism
Several clinical forms of botulism are distinguished according to the mode of acquisition of BoNT and/or neurotoxigenic bacteria. Foodborne botulism occurs after the consumption of food contaminated by
Infant botulism results from the ingestion of
Botulism by intestinal colonization occasionally occurs in adults. Predisposing factors consist in factors that perturb or modify the microbiota composition such as antibiotherapy, intestinal surgery 1 or 2 weeks prior consumption of a food contaminated by
Wound botulism is caused by
Inhalational botulism is very rare. A few cases have been reported in laboratory workers preparing concentrated BoNT by continuous centrifugation and in two patients who inhaled cocaine (review in ). BoNT dissemination by aerosol has been considered as a possible bioterrorist attack.
Iatrogenic botulism is a recent novel form of botulism which develops subsequently to toxin overdoses for a therapeutic or a cosmetic purpose or to a hematological dissemination of toxin at a therapeutic dose.
4.2. Botulism in France
4.2.1. Foodborne botulism
The first case of human botulism was reported in 1875. The disease was very rare until the second war, since the consumption of canned foods was not traditional in France. This not excludes that the disease was underestimated or misdiagnosed. Only 24 cases were recorded from 1875 to 1936 and eight from 1936 to 1940 [126, 127]. In contrast, in the USA where the first industrial canned foods treated by heating were developed, large outbreaks of botulism occurred from 1899 to 1954, 514 outbreaks, 1350 cases including 861 deaths . However, the incidence of botulism was very high in France during the Second World War. About 500 outbreaks and 1000 cases were estimated between 1940 and 1944 . Food deprivation and poor quality of home-made food preservation were the main factors responsible for this high incidence. Type B botulism predominated, and most of the incriminated foods (93%) were from pork meat, notably cured ham .
The incidence of botulism decreased after the Second World War (Figure 2). Albeit no systematic recording of botulism cases was performed during this period, only a few outbreaks were identified, mainly in the Anaerobe Laboratory of Pasteur Institute. During the period 1956–1970, a 22.4 annual mean of botulism cases was observed based mainly on the detection of BoNT and/or
From 1986, human botulism is subjected to mandatory declaration to health authorities and since 1998 botulism declarations are coordinated by the national organism of disease survey InVS (Institut national de Veille Sanitaire called Sante Publique France since 2016). Since 1980, human botulism decreased, but every year, 10–40 cases are recorded in France. Home-made preserved foods are less used but remain traditional in certain areas of France. Type B is predominant, and cured ham and pork meat preparations are the main origin of human botulism [130, 131, 132, 133, 134, 135]. Pork is often a healthy carrier of
Botulism type E is extremely rare in France. An outbreak of botulism type E occurred in 2009 after the consumption of smoked and vacuum-packed fish which was bought a few days ago in Finland. The fish was from Canada and was processed in Finland . In 2010, an unusual case resulted from a ham contaminated with
Two atypical outbreaks of botulism type F occurred in 2014 and 2015. Both were
4.2.2. Infant botulism
Infant botulism is a rare form of botulism in France. Only 15 cases were identified from 2004 to 2016. They resulted from group I
4.2.3. Wound botulism and inhalation botulism
Only one case of wound botulism was identified from 1995 to 2017. In 2008, a patient had an open fracture of the leg abroad and was hospitalized again when back to France for persistent suppuration of the wound. He developed a type B botulism during the course of the second hospitalization . Wound botulism in injection drug users was reported in several European countries and North America, but no such case was reported in France [143, 144]. However, in 2007, two patients who inhaled cocaine developed a botulism type B .
4.2.4. Botulism diversity in France
Albeit botulism is a rare disease, human botulism is identified every year in France. Foodborne botulism is the main form of botulism in France. Historically, home-made cured ham or pork products were the main source of type B botulism. During the recent period, home-made preserved foods including ham are no longer commonly used, but human botulism is still present albeit to a lower extent than in the past. Thereby, the origin of botulism is more diverse including imported products, commercial minimally heated foods, or meals at a restaurant. The diversity of BoNT types and subtypes as well as of the BoNT-producing clostridia reflects the diverse origins of human botulism in France .
BoNTs form a wide diverse family of toxins which target specific neurons, leading to the inhibition of release of neurotransmitters, notably acetylcholine. At least 10 BoNT types and more than 40 subtypes have been identified. All BoNTs retain a common core structure and mode of action which consists in the inhibition of neurotransmitter release, notably acetylcholine, leading to flaccid paralysis. However, they use distinct pathways and distinct intracellular targets to drive the blockade of neurotransmission. Indeed, the distinct BoNT types recognize different neuronal receptors such as different sets of gangliosides and different membrane proteins (SV2 isoforms, synaptotagmin) and target either one of the three SNARE proteins at distinct cleavage sites. In addition, BoNTs are produced by diverse bacterial species, mainly from the