1.C.8 The Botulinum and Tetanus Toxin (BTT) Family
The BTT family consists of a variety of exoneurotoxins from Clostridium botulinum and C. tetani. These include botulinum neurotoxins A-G and tetanus neurotoxin. The 8 botulinum toxins (BoNTs) A-G are sequence and antigenically distinct. BoNTs A B exhibit similar fold and domain association where the translocation domain is flanked on either side by binding and catalytic domains. However, in BoNT E holotoxin, the domain association is different and unique, although the individual domains are similar to those of BoNTs A and B. In BoNT E, both the binding domain and the catalytic domain are on the same side of the translocation domain, and all three have mutual interfaces (Kumaran et al., 2009). These proteins, sometimes called collectively, Clostridial neurotoxins, resemble diphtheria toxin (DT; TC #1.C.7.1.1) in that each of them is secreted as a single polypeptide chain and then cleaved to yield a disulfide-linked heterodimer of a light chain (L; residues 1-447 for botulinum toxin A (BTA)) and a heavy chain (H; residues 448-1295 for BTA). L has pharmacological activity as a protease, a proteolytic blocker of neurotransmitter release, cleaving synaptobrevin. The N- and C-termini of H mediate channel formation for transmembrane transport of BTA L-chain and receptor binding, respectively. HC recognizes both gangliosides and various protein receptors that are required for entry (Chai et al., 2006; Jin et al., 2006). The beltless-translocation domain of Botulinum neurotoxin A embodies a minimum ion-conductive channel (Fischer et al., 2012). Double anchorage to the membrane and an inter-chain disulfide bond are required for the low pH induced entry of tetanus and botulinum neurotoxins into neurons (Pirazzini et al., 2011). VHH antibodies neutralize botulinum neurotoxin E1 by blocking its membrane translocation in host cells (Lam et al. 2020).
There are 10 α-helices and two putative transmembrane α-helical spanners (TMSs) (TH8 and TH9) in the translocation (T) domain of BTT family heavy chains [at positions 626-646 and 655-675 in BTA], and all members of the family exhibit a similar apparent topology. The N-terminal domain of H thus forms a protein translocation pathway. A T-domain fragment, consisting of TH8, TH9 and the interhelical TL5 loop, is sufficient for channel formation. Thus, the DT channel is formed by insertion of this helical hairpin into the membrane. The transport process is probably very similar to that mediated by diphtheria toxin although DT does not show significant sequence similarity with BT or TT. The binding site for chloroquine and related compounds and the influence of binding on properties of the C2II channel have been defined (Neumeyer et al., 2007). Some features of the membrane-bound T (translocation) domain tertiary structure, critical for pore formation, are dependent upon salt concentration (Lai et al., 2010). Swaminathan (2011) has presented an overview of the structure/function relationships, correlating the 3-D structures with biochemical and biophysical analysis of BT.
Clostridia comprise a heterogenous group of environmental bacteria containing 15 pathogenic species, which produce the most potent toxins (Popoff and Bouvet 2013). The origin of toxins is still enigmatic. It is hypothesized that toxins exhibiting an enzymatic activity have derived from hydrolytic enzymes, which are abundantly secreted by these bacteria, and that pore-forming toxins have evolved from an ancestor transmembrane protein. The presence of related toxin genes in distinct Clostridium species and the variability of some toxin genes support horizontal toxin gene transfer and subsequent independent evolution from strain to strain. Clostridium perfringens toxin genes involved in myonecrosis, mainly alpha toxin and perfringolysin genes, are chromosomally located, whereas toxin genes responsible for intestinal and food borne diseases are localized on plasmids except the enterotoxin gene which can be located either on the chromosome or plasmids. The distribution of these plasmids containing one or several toxin genes accounts for the diverse C. perfringens toxinotypes. Clostridium difficile strains show a high genetic variability. But in contrast to C. perfringens, toxin genes are clustered in pathogenicity locus located on chromosome. The presence of related toxin genes in distinct clostridial species like Clostridium sordellii, Clostridium novyi, and C. perfringens supports interspecies mobilization of this locus. The multiple C. difficile toxinotypes based on toxin gene variants possibly reflect strain adaptation to the intestinal environment. Botulinum toxin genes also show a high level of genetic variation. They have a diverse genetic localization including chromosome, plasmid or phage, and are spread in various Clostridium species (Clostridium botulinum groups, Clostridium argentinense, Clostridium butyricum, Clostridium baratii). Exchange of toxin genes not only include transfers between Clostridium species but also between Clostridium and other bacterial species as well as eukaryotic cells as supported by the wide distribution of related pore-forming toxins of the aerolysin family in various clostridial and non-clostridial species, animal, mushroom and plant (Popoff and Bouvet 2013).
Dendrimers are unique highly branched macromolecules with numerous biomedical applications. Förstner et al. 2014 identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. At low μM concentrations, cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, it was shown that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations.
The transport reaction catalyzed by BTT family members is:
L-chain (out) %u2192 L-chain (in)
References:
Botulinum neurotoxin types A-G. Poly(amindo)amine (PAMAM) detrimers block activity (Förstner et al. 2014). BoNTs inhibit synaptic exocytosis; intoxication requires the di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol, colocalizing it with the substrate SNARE proteins (Montal 2009). Botulinum toxin type A inhibits salivary secretion, possibly by alterring RNA synthesis (Mao et al. 2020). pH-dependent structural changes in Botulinum Neurotoxin E have been decumented (Lalaurie et al. 2022).
Bacteria
Botulinum neurotoxin precursor, type A of Clostridium botulinum
Tetanus neurotoxin, TetX, of 1315 aas; secreted (Gupta et al. 2023). Tetanus toxin acts by inhibiting neurotransmitter release. It binds to peripheral neuronal synapses, is internalized, and moves by retrograde transport up the axon into the spinal cord where it can move between postsynaptic and presynaptic neurons. It inhibits neurotransmitter release by acting as a zinc endopeptidase that catalyzes the hydrolysis of the '76-Gln-|-Phe-77' bond of synaptobrevin-2.
Bacteria
Tetanus neurotoxin precursor of Clostridium tetani
Clostridium botulinum neurotoxin (BoNT) type E (The 3d structure is known (Kumaran et al., 2009)). BoNT consists of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). Tan et al. 2023 explored a recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E. Neurotoxicity of L-HN fragments was assessed in mice, and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored. The 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 mug, and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT(A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome- associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter cells via binding to SV2C to efficiently cleave SNAP25. Thus, the EL-HN fragment showed good biological activity and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport (Tan et al. 2023).
Bacteria
BoNTE of Clostridium botulinum (Q00496)
Non-toxic nonhemagglutinin type C of 1196 aas. Assembles with botulinum neurotoxin type C (BoNT/C) and protects it against pH-mediated inactivation or protease activity at pH 2.6 (the pH of the animal gastrointestinal tract) but not at pH 6.0. The non-toxic component is necessary to maintain toxicity.
Nonhemagglutinin type C of Clostridium botulinum C phage (Clostridium botulinum C bacteriophage)