Search

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.

A workshop on the risks associated with animal botulism was held in Maisons-Alfort, France in March 2019. The objectives were to debate the public and animal health risks related to botulism, to disseminate the results obtained from the ANIBOTNET project to other European research teams that were not involved in the project and to other stakeholders, and finally to strengthen and broaden the existing research network on this topic. The workshop was divided into 4 sessions: public and animal health risks associated with botulism, a specific focus on risks associated with agricultural biogas plants, dissemination of ANIBOTNET results, and network development and future perspectives. In all, 58 delegates from 13 countries attended the workshop. A summary of the main topics and discussions during the workshop is presented here.

A workshop on the risks associated with animal botulism was held in Maisons-Alfort, France in March 2019. The objectives were to debate the public and animal health risks related to botulism, to disseminate the results obtained from the ANIBOTNET project to other European research teams that were not involved in the project and to other stakeholders, and finally to strengthen and broaden the existing research network on this topic. The workshop was divided into 4 sessions: public and animal health risks associated with botulism, a specific focus on risks associated with agricultural biogas plants, dissemination of ANIBOTNET results, and network development and future perspectives. In all, 58 delegates from 13 countries attended the workshop. A summary of the main topics and discussions during the workshop is presented here.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and the causative agents of the rare but potentially life-threatening disease botulism. The elaborate mode of action of BoNTs at the molecular level, their exquisite specificity for peripheral motor neurons, and their ability to effectively inhibit neurotransmitter release led to the development of BoNTs into highly valued pharmaceutical products. Both diagnostics of botulism and potency testing of pharmaceutical BoNT preparations still employ the mouse bioassay as “gold standard assay”. This animal experiment can pose a heavy burden on the animal, including a fatal outcome of testing. Additionally, several analytical disadvantages have been described. Consequently, the development of animal replacement methods is a long pursued goal which has been focused mainly on replacement methods for pharmaceutical potency testing so far. However, fundamentally different requirements and challenges apply for diagnostics of botulism and potency testing of BoNT pharmaceuticals, which necessitates the development of different assays tailored for each purpose. Here we review the underlying causes for this intricacy which are rooted in both the biological characteristics of the BoNTs as well as assay specific requirements. We review different functional assays that have been developed to replace the mouse bioassay. Despite significant progress in recent years, further substantial work is needed to pave the way for a fully validated replacement for the mouse bioassay for botulism diagnostics.

Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins.