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Food microbiology : an introduction / Thomas J. Montville and Karl R. Matthews.

Key Points
Botulinum neurotoxins (BoNTs) are produced by neurotoxigenic clostridia and are a diverse group that consists of approximately 40 different BoNT types (and various subtypes), all of which cause persistent paralysis of peripheral nerve terminals — a condition known as botulism.
Recent studies have solved various structures of BoNT complexes, which has provided insights into their modes of entry into the general circulation as well as the ability of these toxins to survive for long periods of time in the
ex vivo
environment.
The molecular basis of the specificity of BoNT binding to nerve terminals is explored, as well as the ensuing cellular events, including toxin endocytosis and the targeting and cleavage of SNARE proteins.
A molecular model for the essential process of membrane translocation of the metalloprotease domain of BoNTs into the neuronal cytosol is presented.
Open questions and future areas of research are outlined with respect to the development of novel therapeutic agents that are based on BoNTs.
Botulinum neurotoxins, which are the most powerful known toxins, are produced by toxigenic clostridia and cause persistent paralysis of peripheral nerve terminals by blocking neurotransmitter release. In this Review, Montecucco and colleagues discuss recent structural and molecular insights into the mechanisms of toxin entry into nerve terminals, membrane translocation and neuroparalysis.
Botulinum neurotoxins (BoNTs) are produced by anaerobic bacteria of the genus
Clostridium
and cause a persistent paralysis of peripheral nerve terminals, which is known as botulism. Neurotoxigenic clostridia belong to six phylogenetically distinct groups and produce more than 40 different BoNT types, which inactivate neurotransmitter release owing to their metalloprotease activity. In this Review, we discuss recent studies that have improved our understanding of the genetics and structure of BoNT complexes. We also describe recent insights into the mechanisms of BoNT entry into the general circulation, neuronal binding, membrane translocation and neuroparalysis.

Nature reviews. Microbiology, 2014-08, Vol.12 (8), p.535-549

Botulinum neurotoxins: genetic, structural and mechanistic insights

BoNT/A1 invades motoneurons by binding to the neuronal receptor SV2. A combination of structural, biophysical and cellular analyses reveal that BoNT/A1 binding and uptake require glycosylation of SV2.
Botulinum neurotoxin serotype A1 (BoNT/A1), a licensed drug widely used for medical and cosmetic applications, exerts its action by invading motoneurons. Here we report a 2.0-Å-resolution crystal structure of the BoNT/A1 receptor-binding domain in complex with its neuronal receptor, glycosylated human SV2C. We found that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—which is conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and for its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an antibotulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications, thereby achieving highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors.

Nature structural & molecular biology, 2016-07, Vol.23 (7), p.656-662

N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A

Group I and
are closely related bacteria responsible for foodborne, infant and wound botulism. A comparative genomic study with 556 highly diverse strains of
Group I and
(including 417 newly sequenced strains) has been carried out to characterise the genetic diversity and spread of these bacteria and their neurotoxin genes. Core genome single-nucleotide polymorphism (SNP) analysis revealed two major lineages;
Group I (most strains possessed botulinum neurotoxin gene(s) of types A, B and/or F) and
(some strains possessed a type B botulinum neurotoxin gene). Both lineages contained strains responsible for foodborne, infant and wound botulism. A new
cluster was identified that included five strains with a gene encoding botulinum neurotoxin sub-type B1. There was significant evidence of horizontal transfer of botulinum neurotoxin genes between distantly related bacteria. Population structure/diversity have been characterised, and novel associations discovered between whole genome lineage, botulinum neurotoxin sub-type variant, epidemiological links to foodborne, infant and wound botulism, and geographic origin. The impact of genomic and physiological variability on the botulism risk has been assessed. The genome sequences are a valuable resource for future research (e.g., pathogen biology, evolution of
and its neurotoxin genes, improved pathogen detection and discrimination), and support enhanced risk assessments and the prevention of botulism.

Toxins, 2020-09, Vol.12 (9), p.586

Diversity of the Genomes and Neurotoxins of Strains of Clostridium botulinum Group I and Clostridium sporogenes Associated with Foodborne, Infant and Wound Botulism

To identify the source of Clostridium botulinum (C. botulinum) involved in a food poisoning case in Kunming and analyze its molecular characteristics.
This study examined samples from a clustered food poisoning incident, including pickled pig liver consumed by three patients, hospital vomit from a deceased patient, and household waste mixed with vomit. Enrichment culture, microscopic examination, and real-time quantitative PCR confirmed the presence of C. botulinum (designated KM001) with type E botulinum toxin. The full-length genomic sequence of this strain was obtained through second and third-generation sequencing, revealing a genome size of 3,713,470 bp. KEGG annotation indicated that 1,840 single genes were assigned to 44 KEGG pathways. Whole-genome sequencing revealed genetic diversity among toxin gene clusters, with 94.76% homology to C. botulinum E3 strain (Alaska E43) and 93.65% homology to C. botulinum B strain (Eklund 17B, NRP). Evolutionary analysis, incorporating complete genomes of foodborne and environmental C. botulinum strains worldwide along with KM001, showed stronger phylogenetic affinity of KM001 to other type E strains.
Overall, this study identified KM001 as the causative agent in a food poisoning incident, marking the first report of type E botulinum toxin poisoning in the region. Genomic analysis revealed the serotype and genetic diversity among toxin gene clusters, providing insights into its gene function, virulence, and evolutionary relationships. Understanding the genetic relationships and evolutionary pathways of different C. botulinum strains is crucial for predicting infection risks and implementing effective control measures.The findings contribute to the documentation of botulinum toxin incidents in Yunnan, China, but do not examine the antimicrobial resistance of C. botulinum or its interactions with other microorganisms; further research is needed to address these aspects.

BMC infectious diseases, 2025-03, Vol.25 (1), p.323-11

Genomic characterisation and traceability analysis of a Clostridium botulinum strain involved in a food poisoning incident

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.

Pathogens and disease, 2018-06, Vol.76 (4)

Genomic insights into the evolution and ecology of botulinum neurotoxins

The neurotoxin formed by
Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of
Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse
Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of
and its neurotoxin genes, dissecting key aspects of
Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

Toxins, 2020-05, Vol.12 (5), p.306

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum ) Associated with Foodborne Botulism and Isolated from the Environment

Clostridium botulinum
(group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure–function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating
C. botulinum
infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure–function link, not only of a molecule but also of the pathogenesis.

Journal of molecular evolution, 2017-04, Vol.84 (4), p.174-186

Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.
Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.
The described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria.

PloS one, 2011-08, Vol.6 (8), p.e23927

Tailored ß-cyclodextrin blocks the translocation pores of binary exotoxins from C. botulinum and C. perfringens and protects cells from intoxication

Botulism is a neuroparalytic disease caused by neurotoxins produced by the bacteria Clostridium botulinum. Botulinum neurotoxins (BoNTs) are among the most potent naturally occurring toxins and are a category A biological threat agent. The 7 toxin serotypes of BoNTs (serotypes A–G) have different toxicities, act through 3 different intracellular protein targets, and exhibit different durations of effect. Botulism may follow ingestion of food contaminated with BoNT, from toxin production of C botulinum present in the intestine or wounds, or from inhalation of aerosolized toxin. Intoxication classically presents as an acute, symmetrical, descending flaccid paralysis. Early diagnosis is important because antitoxin therapy is most effective when administered early. Confirmatory testing of botulism with BoNT assays or C botulinum cultures is time-consuming, and may be insensitive in the diagnosis of inhalational botulism and in as many as 32% of food-borne botulism cases. Therefore, the decision to initiate botulinum antitoxin therapy is primarily based on symptoms and physical examination findings that are consistent with botulism, with support of epidemiological history and electrophysiological testing. Modern clinical practice and antitoxin treatment has reduced botulism mortality rates from ∼60% to ≤10%. The pentavalent botulinum toxoid is an investigational product and has been used for more than 45 years in at-risk laboratory workers to protect against toxin serotypes A to E. Due to declining immunogenicity and potency of the pentavalent botulinum toxoid, novel vaccine candidates are being developed. (Disaster Med Public Health Preparedness. 2007;1:122–134)

Disaster medicine and public health preparedness, 2007-11, Vol.1 (2), p.122-134

Botulism: Cause, Effects, Diagnosis, Clinical and Laboratory Identification, and Treatment Modalities

Highlights•Replicon vaccines for BoNTs afford stronger potency than conventional DNA vectors. •Dual-expressing replicon vaccines effectively protection against two BoNT serotypes. •Two dual-expressing replicon tetravalent vaccines protect against four BoNT serotypes. •We describe a potent strategy to develop multivalent vaccines using replicon vectors.

Vaccine, 2013-05, Vol.31 (20), p.2427-2432

Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors

Botulism is a potentially fatal infection characterized by progressive muscle weakness, bulbar paralysis, constipation and other autonomic dysfunctions. A recent report suggested that cancer chemotherapy might increase the risk for the intestinal toxemia botulism in both adults and children.
We report a 5-year-old boy, who developed general muscle weakness, constipation, ptosis and mydriasis during the third induction therapy for relapsed acute myeloid leukemia. He had recent histories of multiple antibiotic therapy for bacteremia and intake of well water at home. Repeated bacterial cultures identified Clostridium botulinum producing botulinum neurotoxin A. Botulinum toxin A was isolated from his stools at 17, 21, and 23 days after the onset. Symptoms were self-limiting, and were fully recovered without anti-botulinum toxin globulin therapy.
This is the second report of a pediatric case with cancer chemotherapy-associated intestinal toxemia botulism. Our case provides further evidence that the immunocompromised status due to anti-cancer treatments increases the risk for the development of botulism at all ages in childhood.

Annals of clinical microbiology and antimicrobials, 2017-09, Vol.16 (1), p.61-61, Article 61

A childhood-onset intestinal toxemia botulism during chemotherapy for relapsed acute leukemia

Journal of neurology, neurosurgery and psychiatry, 2004-09, Vol.75 (suppl 3), p.iii35-iii39

Clostridial neurotoxins

Equine grass sickness (EGS) occurs mainly in Great Britain, but has once been reported in Hungary. The stud which was affected by EGS in 2001 had no new cases until 2009/10, when 11 of 60 and five of 12 one- to three-year-old colts died or were euthanased due to EGS. Following a few hours in the high-risk field during the winter of 2010/11 further four cases of acute EGS were noted among these horses. The affected horses showed somewhat different clinical signs compared with the cases reported in Great Britain. Histopathological findings in these horses were consistent with EGS. In most examined cases carbofuran, a carbamate was found in the liver by toxicological examination, and it is postulated that carbofuran may influence the immune system and therefore predispose the horses to develop EGS. Carbamates are thought to cause a delayed neurotoxicity in human beings. Further studies are needed to clarify the potential role of carbamates in EGS.

Veterinary record, 2012-01, Vol.170 (3), p.75-75

Outbreaks of equine grass sickness in Hungary

Botulism is an acute form of poisoning caused by one of four types (A, B, E, F) toxins produced by Clostridium botulinum, ananaerobic, spore forming bacillus. Usually diagnosis of botulism is considered in patients with predominant motor symptoms: muscle weakness with intact sensation and preserved mental function.
We report a case of 56-year-old Caucasian female with a history of arterial hypertension, who presented with acute respiratory failure and bilateral ptosis misdiagnosed as brainstem ischemia. She had severe external and internal ophtalmoplegia, and autonomic dysfunction with neither motor nor sensory symptoms from upper and lower limbs. Diagnosis of botulinum toxin poisoning was made and confirmed by serum antibody testing in the mouse inoculation test.
Ophtalmoplegia, autonomic dysfunction and respiratory failure can be caused by botulism. Early treatment and intensive care is essential for survival and recovery. The electrophysiological tests are crucial to correct and rapid diagnosis. Botulism (especially type B) should be considered in any case of acute or predominant isolated autonomic dysfunction.

BMC research notes, 2013-10, Vol.6 (1), p.438-438, Article 438

Long lasting dysautonomia due to botulinum toxin B poisoning: clinical-laboratory follow up and difficulties in initial diagnosis

Science (American Association for the Advancement of Science), 2015-02, Vol.347 (6225), p.934-935

Biosecurity. As new botulism threat implodes, more questions

Several varieties of birds are affected by type C botulism. We conducted neutralization tests of culture supernatants of isolates from cases of avian botulism. Whereas the toxin produced by isolates derived from mammalian botulism was neutralized only with type C antitoxin, the toxins of all isolates related to avian botulism were neutralized with both type C and D antitoxins. An analysis of nucleotide sequences with several strains revealed that the neurotoxin gene in the isolates from avian botulism comprises two thirds of the type C neurotoxin gene and one third of the type D neurotoxin gene. This indicates that the neurotoxin of avian isolates is a mosaic of type C and D neurotoxins. We prepared three sets of primers to differentiate the gene for the mosaic form from the conserved genes of type C and D neurotoxins. The results of polymerase chain reaction with these primers indicated that all avian botulism–related isolates and specimens possess the gene for the mosaic form of the neurotoxin. The toxins purified from avian and mammalian isolates exhibited the same degree of lethality in mice, but the former showed greater toxicity to chickens than the latter. These results indicate that the mosaic neurotoxin is probably a pathogenic agent causing some forms of avian botulism.

Avian diseases, 2005-09, Vol.49 (3), p.376-381

Characterization of the Neurotoxin Produced by Isolates Associated with Avian Botulism

We have reviewed the information about epitopes of immunological interest from Clostridium botulinum and Bacillus anthracis, by mining the Immune Epitope Database and Analysis Resource. For both pathogens, the vast majority of epitopes reported to date are derived from a single protein: the protective antigen of B. anthracis and the neurotoxin type A of C. botulinum. A detailed analysis of the data was performed to characterize the function, localization and conservancy of epitopes identified as neutralizing and/or protective. In order to broaden the scope of this analysis, we have also included data describing immune responses against defined fragments (over 50 amino acids long) of the relevant antigens. The scarce information on T-cell determinants and on epitopes from other antigens besides the toxins, highlights a gap in our knowledge and identifies areas for future research. Despite this, several distinct structures at the epitope and fragment level are described herein, which could be potential additions to future vaccines or targets of novel immunotherapeutics and diagnostic reagents.

Expert review of vaccines, 2008-02, Vol.7 (1), p.55-74

Analysis of epitope information related to Bacillus anthracis and Clostridium botulinum

Equine grass sickness (EGS) is a largely fatal, pasture-associated dysautonomia. Although the aetiology of this disease is unknown, there is increasing evidence that Clostridium botulinum type C plays an important role in this condition. The disease is widespread in the United Kingdom, with the highest incidence believed to occur in Scotland. EGS also shows strong seasonal variation (most cases are reported between April and July). Data from histologically confirmed cases of EGS from England and Wales in 1999 and 2000 were collected from UK veterinary diagnostic centres. The data did not represent a complete census of cases, and the proportion of all cases reported to the centres would have varied in space and, independently, in time. We consider the variable reporting of this condition and the appropriateness of the space–time K-function when exploring the spatial-temporal properties of a ‘thinned’ point process. We conclude that such position-dependent under-reporting of EGS does not invalidate the Monte Carlo test for space–time interaction, and find strong evidence for space–time clustering of EGS cases (P<0·001). This may be attributed to contagious or other spatially and temporally localized processes such as local climate and/or pasture management practices.

Epidemiology and infection, 2005-04, Vol.133 (2), p.343-348

Clustering of equine grass sickness cases in the United Kingdom: a study considering the effect of position-dependent reporting on the space–time K-function

Wound botulism is a rare infectious disease that is becoming a frequent complication of parental drug use. Diagnosis is often difficult and based on clinical suspicion. We report the first Italian case of wound botulism due to intramuscular heroin injection in a 48-year-old man with an acute onset of slurred speech and dysphagia. The most considerable finding of electrophysiological study was the reduction in amplitude of compound muscle action potential which should be considered a useful initial electrodiagnostic sign in the clinical context of botulism. Alerting clinicians to botulism is crucial for a rapid diagnosis and appropriate treatment and thus decreasing mortality and complications.