A Family Outbreak of Type E Botulism Caused by Contaminated Vacuum-Packed Ambient-Stored Chili Chicken Feet in Zhangjiakou, China.

The epidemiological investigation and laboratory-based confirmation were performed on samples from a family botulism outbreak in Zhangjiakou, Hebei province, China. Forty-four samples, including 14 samples (leftover food, and swabs taken of both food packaging bags and dishes, and serum and vomitus of the victims) related to outbreak and 30 causative food products after outbreak, were collected and analyzed. Isolation, bacterial identification, toxin detection, and whole-genome sequencing of Clostridium spp. cultured from the latter samples and animal assays were performed. Mice injected with the cultures of the leftover chili chicken feet, together with the inner layer of its packaging bag, the plate for serving it, and supernatant of two patients' serum that demonstrated the typical signs of botulism. The polyvalent anti-botulinum neurotoxin (BoNT) and the monovalent anti-BoNT/E exhibited protective effects when administered to mice. Three Clostridium botulinum cultures were obtained and verified to be positive for BoNT/E. The whole genome analysis of the isolates revealed that the classic bont/e gene orfX cluster was found to be located on the chromosomes of all three isolates. Single nucleotide polymorphism analysis suggested that these might be from the same source. Our findings indicated that this botulism outbreak occurred following the ingestion of vacuum-packed chili chicken feet contaminated with BoNT/E produced by C. botulinum.

Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E.

The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228-1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides.

[Foodborne botulism - a re-emerging public health challenge]. / Alimentárny botulizmus - staronová výzva pre verejné zdravotníctvo.

Human foodborne botulism is an intoxication caused by ingestion of botulinum neurotoxins (BoNT) of serotypes A, B, E, and rarely also serotype F, produced in contaminated food by anaerobic bacteria Clostridium botulinum group I, group II, or by toxigenic strains of C. butyricum and C. baratii. BoNT-producing Clostridia are ubiquitously distributed in the environment and, under suitable conditions, they can enter the food chain, proliferate and produce BoNT in a variety of foods. In the past, the risk of foodborne botulism was primarily associated with homemade canned foods; however, the epidemiological importance of commercial and restaurant food is increasing nowadays. In this article, we review the public health aspects of foodborne botulism, including its clinical, epidemiological and laboratory diagnosis and discuss potential risks associated with minimally heated, vacuum or modified atmosphere-packed, ready-to-eat foods of extended durability.

Identification of small molecule inhibitors of botulinum neurotoxin serotype E via footprint similarity.

Botulinum neurotoxins (BoNT) are among the most poisonous substances known, and of the 7 serotypes (A-G) identified thus far at least 4 can cause death in humans. The goal of this work was identification of inhibitors that specifically target the light chain catalytic site of the highly pathogenic but lesser-studied E serotype (BoNT/E). Large-scale computational screening, employing the program DOCK, was used to perform atomic-level docking of 1.4 million small molecules to prioritize those making favorable interactions with the BoNT/E site. In particular, 'footprint similarity' (FPS) scoring was used to identify compounds that could potentially mimic features on the known substrate tetrapeptide RIME. Among 92 compounds purchased and experimentally tested, compound C562-1101 emerged as the most promising hit with an apparent IC50 value three-fold more potent than that of the first reported BoNT/E small molecule inhibitor NSC-77053. Additional analysis showed the predicted binding pose of C562-1101 was geometrically and energetically stable over an ensemble of structures generated by molecular dynamic simulations and that many of the intended interactions seen with RIME were maintained. Several analogs were also computationally designed and predicted to have further molecular mimicry thereby demonstrating the potential utility of footprint-based scoring protocols to help guide hit refinement.

Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library.

AIMS: To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR. METHODS AND RESULTS: The gene coding for specific VHH derived from the camel immunized with binding domain of botulinum neurotoxin E (BoNT/E) was amplified by error-prone PCR. Several biopanning rounds were used to screen the phage-displaying BoNT/E Hc nanobodies. The final nanobody, SHMR4, with increased affinity recognized BoNT/E toxin with no cross-reactivity with other antigens especially with related BoNT toxins. CONCLUSIONS: The constructed nanobody could be a suitable candidate for VHH-based biosensor production to detect the Clostridium botulinum type E. SIGNIFICANCE AND IMPACT OF THE STUDY: Diagnosis and treatment of botulinum neurotoxins are important. Generation of high-affinity antibodies based on the construction of secondary libraries using affinity maturation step leads to the development of reagents for precise diagnosis and therapy.

Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM.

Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.

Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E.

Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A-G). Most foodborne intoxications are caused by serotypes BoNT/A and BoNT/B. BoNT/E outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of BoNT/E3. Monoclonal antibodies (mAbs) were generated against BoNT/E3 by immunizing with recombinant peptide fragments of the light and heavy chains of BoNT/E3. In all, 12 mAbs where characterized for binding to both the recombinant peptides and holotoxin, as well as their performance in Western blots and sandwich ELISAs. The most sensitive sandwich assay, using different mAbs for capture and detection, exhibited a limit of detection of 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. By employing two different mAbs for capture and detection, a more standardized sandwich assay was constructed. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals.

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E.

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.