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Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)