Genomic insights into the evolution and ecology of botulinum neurotoxins

Abstract

Clostridial neurotoxins, which include botulinum neurotoxins (BoNTs) and tetanus neurotoxins, have evolved a remarkably sophisticated structure and molecular mechanism fine-tuned for the targeting and cleavage of vertebrate neuron substrates leading to muscular paralysis. How and why did this toxin evolve? From which ancestral proteins are BoNTs derived? And what is, or was, the primary ecological role of BoNTs in the environment? In this article, we examine these questions in light of recent studies identifying homologs of BoNTs in the genomes of non-clostridial bacteria, including Weissella, Enterococcus and Chryseobacterium. Genomic and phylogenetic analysis of these more distantly related toxins suggests that they are derived from ancient toxin lineages that predate the evolution of BoNTs and are not limited to the Clostridium genus. We propose that BoNTs have therefore evolved from a precursor family of BoNT-like toxins, and ultimately from non-neurospecific toxins that cleaved different substrates (possibly non-neuronal SNAREs). Comparison of BoNTs with these related toxins reveals several unique molecular features that underlie the evolution of BoNT's unique function, including functional shifts involving all four domains, and gain of the BoNT gene cluster associated proteins. BoNTs then diversified to produce the existing serotypes, including TeNT, and underwent repeated substrate shifts from ancestral VAMP2 specificity to SNAP25 specificity at least three times in their history. Finally, similar to previous proposals, we suggest that one ecological role of BoNTs could be to create a paralytic phase in vertebrate decomposition, which provides a competitive advantage for necrophagous scavengers that in turn facilitate the spread of Clostridium botulinum and its toxin.

Variations in the Botulinum Neurotoxin Binding Domain and the Potential for Novel Therapeutics

“…It is not a trivial task to determine how BoNTs bind to their receptors on neuronal cell membranes, especially when trying to replicate the conditions in vivo. With the recent discovery of new BoNTs and BoNT-like molecules in other bacterial species, this raises questions regarding the evolution of the bont gene cluster, their ability to be transferred between species, the potential implications for biosafety, and the need for an agreed-upon consistent naming convention to avoid confusion and ambiguity [ 132 , 133 ]. Fast characterisation and the generation of neutralising antibodies against these novel toxins is required.…”

The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs

“…TeNT and BoNT are also commonly indicated as clostridial neurotoxins, because until few years ago, they were known to be produced only by bacteria of the genus Clostridium . This indication is no longer correct as toxins with very similar structure and enzymatic target were recently discovered using bioinformatic methods in non‐clostridial bacterial species (Brunt, Carter, Stringer, & Peck, ; Doxey, Mansfield, & Montecucco, ; Mansfield, Adams, & Doxey, ; Mansfield & Doxey, ; Mansfield et al, ; Zhang et al, ; Zornetta et al, ). However, these “bioinformatics” toxins have not been yet associated to diseases nor it is known if they are produced at all in the natural environment (Doxey et al, ).…”

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

“…BoNTs are produced primarily by the Gram-positive, anaerobic, spore-forming bacterial species Clostridium botulinum [31,[33][34][35]. In addition, genetically related homologs of BoNTs have also been identified in more distantly related genera, including Enterococcus, Weissella, and Chryseobacterium [17,[36][37][38][39][40][41][42]. However, while the catalytic activity of these BoNT-like LCs on SNARE proteins has been shown, there are currently no indications that these homologs act as vertebrate neurotoxins, and further research will be required to determine potential toxicity, target hosts, and biologic function.…”

“…Interestingly, recent research of novel BoNTs has found that BoNT/F5 and BoNT/FA are an exception within the F subtype LCs in that they cleave VAMP1/2 (vesicle-associated membrane protein) at unique sites, which are distinct from the cleavage sites of other type F LCs [72,73]. Interestingly, the novel putative BoNT/X, as well as BoNT homologs from organisms other than clostridia such as BoNT/Wo and BoNT/En, have been found to have unique neuronal and in some cases non-neuronal SNARE cleavage targets and sites [17,32,[36][37][38][39][40][41][42]. It is currently unknown whether novel or as yet uncharacterized BoNTs may have unique SNARE cleavage targets, which is an important consideration when using a BoNT detection method that relies on detection of specific SNARE cleavage.…”

“…BoNTs also have been exceedingly useful tools in neuroscience research due to their ability to completely block neurotransmitter release by neurons, affecting many physiological processes. Finally, the large family of BoNTs continues to expand with the discovery of new subtypes and chimeric toxins, and much remains to be learned about the biology and evolutionary significance of these novel toxins [14][15][16][17]. Detection of BoNTs is essential for all facets of BoNTs, including safety, the morbidity of botulism, regulatory decisions and actions, pharmaceutical development, and use of BoNTs, and basic research.…”