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. H_C 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).

The transport reaction catalyzed by BTT family members is:

L-chain (out) %u2192 L-chain (in)