Molecular Characterization of Clostridium botulinum Harboring the bont/B7 Gene.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious disease that can result in death if not treated. Infant botulism occurs when C. botulinum colonizes the intestinal tract of infants and produces BoNT. It has been proposed that infants under the age of 1 year are uniquely susceptible to colonization by C. botulinum as their intestinal microbiota is not fully developed and provides little competition, allowing C. botulinum to thrive and produce BoNT in the gut. There are seven well-characterized serotypes (A-G) of BoNT identified by the ability of specific antitoxins to neutralize BoNTs. Molecular technology has allowed researchers to narrow these further into subtypes based on nucleic acid sequences of the botulinum toxin (bont) gene. One of the most recently recognized subtypes for bont/B is subtype bont/B7. We identified through whole genome sequencing five C. botulinum isolates harboring bont/B7 from CDC's strain collection, including patient isolates and an epidemiologically linked isolate from an opened infant formula container. In this study, we report the results of whole genome sequencing analysis of these C. botulinum subtype bont/B7 isolates. Average nucleotide identity and high quality single nucleotide polymorphism (hqSNP) analysis resulted in two major clades. The epidemiologically linked isolates differed from each other by 2-6 hqSNPs, and this clade separated from the other isolates by 95-119 hqSNPs, corroborating available epidemiological evidence.

[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.

Exploring the genetic background of the botulism neurotoxin BoNT/B2 in Spain.

To determine whether the neurotoxin BoNT/B2 causing botulism in Spain is clonal, the genetic diversity and phylogenetic relationships of Clostridium botulinum from food-borne episodes and infant cases of the condition were explored. The botulinum toxin gene (bont) subtype, the variable region of the flagellin gene (flaVR), and a seven-gene multi-locus sequence type were examined by sequencing 37 BoNT-positive cultures obtained over the period 2010 to 2022. Out of 37 botulism events, 16 food-borne episodes and 16 infant cases were associated with bont/b2. Eight bont/b2 alleles were detected [nucleotide distance range 0.0259-0.415%, Hunter and Gaston discrimination index (HGDI) 0.71]. The most common bont/b2 allele corresponded to that of strain Prevot 25 NCASE and its single and double locus variations (87.5%). Four known flaVR types were identified (HGDI 0.79), along with one previously unknown (flaVR-15). Sixteen sequence types (STs) (HGDI 0.89) were recorded including seven new STs (ST164-ST170; 10 new alleles) and five new STs (ST171-ST175; with new allele combinations) were also noted. Correlations among some STs and flaVR types were seen. Overall, the present results show that the combined analysis of bont/b2-flaVR-ST at the nucleotide level could be used to track botulism events in Spain. The neurotoxin BoNT/B2 has largely been responsible for human botulism in Spain. The polymorphism analysis of bont/b2, flaVR typing, and sequence type determinations, revealed a wide variety of clones to be responsible for human botulism, ruling out a common source of acquisition. IMPORTANCE Botulism, a potentially fatal disease, is classically characterized by a symmetrical descending flaccid paralysis, which if left untreated can lead to respiratory failure and death. Botulinum neurotoxin (BoNT), produced by certain species of Clostridium, is the most potent biological toxin known, and the direct cause of botulism. This study characterizes the acquisition in Spain of two forms of botulism, i.e., food-borne and infant botulism, which are largely caused by the main neurotoxin BoNT/B2. Polymorphism analysis of the bont/b2 gene, typing of the flagellin variable region sequence (flaVR), and multilocus sequence typing, were used to explore the genetic background of Clostridium botulinum group I. To our knowledge, this is the first phylogenetic and typing study of botulism undertaken in Spain.

Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay.

Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.

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.

Clostridium botulinum Type B Isolated From a Wound Botulism Case Due to Injection Drug Use Resembles Other Local Strains Originating From Hawaii.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which can lead to death if untreated. In the United States, over 90% of wound botulism cases are associated with injection drug use of black tar heroin. We sought to determine the phylogenetic relatedness of C. botulinum isolated from an injection drug use wound botulism case and isolates from endogenous infant botulism cases in Hawaii. Nineteen C. botulinum type B isolates from Hawaii and one type B isolate from California were analyzed by whole-genome sequencing. The botulinum toxin gene (bont) subtype was determined using CLC Genomics Workbench, and the seven-gene multi-locus sequence type (MLST) was identified by querying PubMLST. Mashtree and pairwise average nucleotide identity were used to find nearest neighbors, and Lyve-SET approximated a phylogeny. Eighteen of the isolates harbored the bont/B5 gene: of those, 17 were classified as sequence type ST36 and one was classified as ST104. A single isolate from Hawaii harbored bont/B1 and was determined to belong to ST110, and the isolate from California harbored bont/B1 and belonged to ST30. A tree constructed with Lyve-SET showed a high degree of homology among all the Hawaiian C. botulinum isolates that harbor the bont/B5 gene. Our results indicate that the bont/B-expressing isolates recovered from Hawaii are closely related to each other, suggesting local contamination of the drug paraphernalia or the wound itself with spores rather than contamination of the drug at manufacture or during transport. These findings may assist in identifying interventions to decrease wound botulism among persons who inject drugs.

Structural and Biochemical Characterization of Botulinum Neurotoxin Subtype B2 Binding to Its Receptors.

Botulinum neurotoxins (BoNTs) can be used therapeutically to treat a wide range of neuromuscular and neurological conditions. A collection of natural BoNT variants exists which can be classified into serologically distinct serotypes (BoNT/B), and further divided into subtypes (BoNT/B1, B2, …). BoNT subtypes share a high degree of sequence identity within the same serotype yet can display large variation in toxicity. One such example is BoNT/B2, which was isolated from Clostridium botulinum strain 111 in a clinical case of botulism, and presents a 10-fold lower toxicity than BoNT/B1. In an effort to understand the molecular mechanisms behind this difference in potency, we here present the crystal structures of BoNT/B2 in complex with the ganglioside receptor GD1a, and with the human synaptotagmin I protein receptor. We show, using receptor-binding assays, that BoNT/B2 has a slightly higher affinity for GD1a than BoNT/B1, and confirm its considerably weaker affinity for its protein receptors. Although the overall receptor-binding mechanism is conserved for both receptors, structural analysis suggests the lower affinity of BoNT/B2 is the result of key substitutions, where hydrophobic interactions important for synaptotagmin-binding are replaced by polar residues. This study provides a template to drive the development of future BoNT therapeutic molecules centered on assessing the natural subtype variations in receptor-binding that appears to be one of the principal stages driving toxicity.

The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity.

The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain HN, and the C-terminal receptor binding domain HCC are largely resolved, but that of the HCN domain sandwiched between HN and HCC has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their HCN domains or swapping HCN domains between each other. Both deletion and replacement of TeNT HCN domain by HCNA and HCNB reduced the biological activity similarly, by ~95%, whereas BoNT/A and B deletion mutants displayed >500-fold reduced activity in the mouse phrenic nerve hemidiaphragm assay. Swapping HCN domains between BoNT/A and B hardly impaired their biological activity, but substitution with HCNT did. Binding assays revealed that in the absence of HCN, not all receptor binding sites are equally well accessible. In conclusion, the presence of HCN is vital for CNTs to exert their neurotoxicity. Although structurally similar, the HCN domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that HCNT plays a different role in the intoxication mechanism of TeNT.

Clinical differences between botulinum neurotoxin type A and B.

In humans, the therapeutic use of botulinum neurotoxin A (BoNT/A) is well recognized and continuously expanding. Four BoNTs are widely available for clinical practice: three are serotype A and one is serotype B: onabotulinumtoxinA (A/Ona), abobotulinumtoxinA (A/Abo) and incobotulinumtoxinA (A/Inco), rimabotulinumtoxinB (B/Rima). A/Abo, A/Inco, A/Ona and B/Rima are all licensed worldwide for cervical dystonia. In addition, the three BoNT/A products are approved for blepharospasm and focal dystonias, spasticity, hemifacial spasm, hyperhidrosis and facial lines, with remarkable regional differences. These toxin brands differ for specific activity, packaging, constituents, excipient, and storage. Comparative literature assessing the relative safety and efficacy of different BoNT products is limited, most data come from reports on small samples, and only a few studies meet criteria of evidence-based medicine. One study compared the effects of BoNT/A and BoNT/B on muscle activity of healthy volunteers, showing similar neurophysiological effects with a dose ratio of 1:100. In cervical dystonia, when comparing the effects of BoNT/A and BoNT/B, results are more variable, some studies reporting roughly similar peak effect and overall duration (at a ratio of 1:66, others reporting substantially shorter duration of BoNT/B than BoNT/A (at a ratio 1/24). Although the results of clinical studies are difficult to compare for methodological differences (dose ratio, study design, outcome measures), it is widely accepted that: BoNT/B is clinically effective using appropriate doses as BoNT/A (1:40-50), injections are generally more painful, in most of the studies on muscular conditions, efficacy is shorter, and immunogenicity higher. Since the earliest clinical trials, it has been reported that autonomic side effects are more frequent after BoNT/B injections, and this observation encouraged the use of BoNT/B for sialorrhea, hyperhidrosis and other non-motor symptoms. In these indications the efficacy of toxins A and B are comparable and dose ratio is 1:25-30.

Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which suggest that they may be well tolerated in potential clinical development.

Engineering Clostridia Neurotoxins with elevated catalytic activity.

BoNT/B and TeNT cleave substrate VAMP2 at the same scissile bond, yet these two toxins showed different efficiency on substrate hydrolysis and had different requirements for the recognition of P2' site of VAMP2, E(78). These differences may be due to their different composition of their substrate recognition pockets in the active site. Swapping of LC/T S1' pocket residue, L(230), with the corresponding isoleucine in LC/B increased LC/T activity by ∼25 fold, while swapping of LC/B S1' pocket residue, S(201), with the corresponding proline in LC/T increased LC/B activity by ∼10 fold. Optimization of both S1 and S1' pocket residues of LC/T, LC/T (K(168)E, L(230)I) elevated LC/T activity by more than 100-fold. The highly active LC/T derivative engineered in this study has the potential to be used as a more effective tool to study mechanisms of exocytosis in central neuron. The LC/B derivative with elevated activity has the potential to be developed into novel therapy to minimize the impact of immunoresistance during BoNT/B therapy.

Analgesic effects of intra-articular botulinum toxin Type B in a murine model of chronic degenerative knee arthritis pain.

OBJECTIVE: To evaluate the analgesic effectiveness of intra-articular botulinum toxin Type B (BoNT/B) in a murine model of chronic degenerative arthritis pain. METHODS AND MATERIALS: Chronic arthritis was produced in adult C57Bl6 mice by intra-articular injection of Type IV collagenase into the left knee. Following induction of arthritis, the treatment group received intra-articular BoNT/B. Arthritic control groups were treated with intra-articular normal saline or sham injections. Pain behavior testing was performed prior to arthritis, after induction of arthritis, and following treatments. Pain behavior measures included analysis of gait impairment (spontaneous pain behavior) and joint tenderness evaluation (evoked pain response). Strength was measured as ability to grasp and cling. RESULTS: Visual gait analysis showed significant impairment of gait in arthritic mice that improved 43% after intra-articular BoNT/B, demonstrating a substantial articular analgesic effect. Joint tenderness, measured with evoked pain response scores, increased with arthritis induction and decreased 49.5% after intra-articular BoNT/B treatment. No improvement in visual gait scores or decrease in evoked pain response scores were found in the control groups receiving intra-articular normal saline or sham injections. Intra-articular BoNT/B was safe, and no systemic effects or limb weakness was noted. CONCLUSIONS: This study is the first report of intra-articular BoNT/B for analgesia in a murine model of arthritis pain. The results of this study validate prior work using intra-articular neurotoxins in murine models. Our findings show chronic degenerative arthritis pain can be quantitated in a murine model by measuring gait impairment using visual gait analysis scores (spontaneous pain behavior) and joint tenderness scores (evoked pain responses). Reduction of joint pain seen in this study is consistent with our hypothesis of inhibition of release of pain mediators by intra-articular BoNT/B, supporting further investigation of this novel approach to treatment of arthritis pain with intra-articular neurotoxins.