*3.4.1 Botulinum neurotoxins*

This neurotoxin is produced by anaerobic, spore forming, gram-positive bacteria belong to genus Clostridium having more than 150 bacterial species. Botulinum neutoxin (BoNTs) consist of 7 serotypes (A-G) and possess more than 40 subtypes.

Botulism disease is caused by Botulinum neurotoxin and A, B, C, E and F serotypes are responsible for causing this disease in human beings. In 2013 a new toxin was reported in human infant. This toxin was known as BoNT/H, BoNT/FA and BoNT/ HA produces by bivalent *Clostridium botulinum* bacterial strain. Genetically it was analyzed that it has a toxin genes BoNT/H shares ≈ 84% resemblance with BoNT/A1 in its binding site, ≈ 80% with BoNT/F5 in its catalytic domain and exhibits a translocation domain identical to BoNT/F1. Further analyses revealed that this BoNT/H toxin neutralizes by available antisera. Though, its unusual toxicological effects, shows lower potency and development of botulism symptoms as compared to primary BoNTs. Various studies describe that there are some resemblances between BoNT/H (i.e., BoNT/FA or BoNT/HA) and BoNT/F or BoNT/A and this creates unanimity. about the position of the BoNT/H [41, 42].

The diversity of Botulinum neurotoxins was further explored by the using modern techniques such as sequencing and genomic analysis, bioinformatics and data-mining tools. BoNTs and its genes have been reported in *Enterococcus feacium* or Weisseria oryzae. These bacterial strains can produce BoNT toxins that depict same characteristics in the multidomain organization of BoNTs but with different toxicological features, specificity or operation principles [42]. BoNT/X is the first serotype of BoNTs identified by bioinformatics techniques and genomic sequencing. Toxin was identified in a *Clostridium botulinum* strain, which displayed the typical BoNTs characteristics including the residues forming a ganglioside binding pocket and metalloprotease consensus sequence or interchain disulphide bond. A significant trait of BoNT/X is its ability to cleave VAMP4 and atypical SNARE Ykt6. A new serotype, BoNT/X, BoNT/ En and Weissella oryzae BoNT-like toxin (also known as BoNT/Wo) have been discovered in recent years. The bioinformatics analysis of bacterium *Weisella oryzae* has led to the recognition of an open reading frame 1, which has a strong sequence resemblance with both genes, but that does not contain the additional genes usually associated within the locus in *Clostridia.* The sequence similarity between BoNT/Wo and other BoNTs is ~14–16%. Moreover, the two cysteines that are part of essential inter-chain disulfide bond in BoNTs are not conserved in BoNT/Wo, depicting a different mode of action. A novel BoNT-like gene was identified in the genome of *Enterococcus faecium.* This BoNT called as BoNT/En composed of distinctive botulinum neurotoxins domain architecture, disulfide bond residues and conservation of the zinc peptidase HExxH motif. BoNT/En describes 29–38.7% similarity with the other BoNTs [43, 44].

Botulinum toxin mainly composed of metalloproteins (zinc-dependent endopeptidases). This toxin having single polypeptides cut with the help of clostridial or host proteases to its active form. The C-terminal heavy chain constitutes the binding and translocation domains of 50 kDa each, and is joined by a single disulfide bridge to the catalytic light chain making the N-terminal part [45]. This protein possesses three domains responsible for its biological functions. The binding domain is responsible for combining the toxin to the receptor on the surface of the cell membrane of the target the nerve cells. The translocation domain is responsible for the transport of toxin through the plasma membrane into the cell, while the enzymatic domain (with proteolytic activity) causes the detachment of peptide bonds in the SNARE transmembrane protein (SNAP-25, VAMP-1/2, syntaxin-1/2) that are integral to vesicular trafficking and neurotransmitters release. Depending on the type of toxin, the proteolytic effect is directed at different SNARE proteins. BoNT A and BoNT E cause hydrolysis SNAP-25 membrane protein. Type B, F and G carry out hydrolysis VAMP-1/2, while BoNT C conducts hydrolysis SNAP 25 and syntaxin-1/2 [46, 47]. The procedure of the toxic action of botulin toxin stops the release of neurotransmitters,
