Potency Evaluations of Recombinant Botulinum Neurotoxin A1 Mutants Designed to Reduce Toxicity.

Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2-3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold-12.5-million-fold) versus wild-type BoNT/A1, leading to their current exclusion from the Federal Select Agent list. In this study, we added four additional mutations in the receptor-binding domain, translocation domain, and enzymatic cleft to further decrease toxicity, creating 7M rBoNT/A1. Due to poor expression in E. coli, 7M rBoNT/A1 was produced in an endogenous C. botulinum expression system. This protein had higher residual toxicity (LD50: 280 ng/mouse) than previously reported for the catalytically inactive rBoNT/A1 containing only three of the mutations (>10 µg/mouse). To investigate this discrepancy, several additional rBoNT/A1 constructs containing individual sets of amino acid substitutions from 7M rBoNT/A1 and related mutations were also endogenously produced. Similarly to endogenously produced 7M rBoNT/A1, all of the endogenously produced mutants had ~100-1000-fold greater toxicity than what was reported for their original heterologous host counterparts. A combination of mutations in multiple functional domains resulted in a greater but not multiplicative reduction in toxicity. This report demonstrates the impact of production systems on residual toxicity of genetically inactivated rBoNTs.

Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy.

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.

Tetanus toxin and botulinum neurotoxin-derived fusion molecules are effective bivalent vaccines.

Tetanus toxin (TeNT) and botulinum neurotoxins (BoNTs) are neuroprotein toxins, with the latter being the most toxic known protein. They are structurally similar and contain three functional domains: an N-terminal catalytic domain (light chain), an internal heavy-chain translocation domain (HN domain), and a C-terminal heavy chain receptor binding domain (Hc domain or RBD). In this study, fusion functional domain molecules consisting of the TeNT RBD (THc) and the BoNT/A RBD (AHc) (i.e., THc-Linker-AHc and AHc-Linker-THc) were designed, prepared, and identified. The interaction of each Hc domain and the ganglioside receptor (GT1b) or the receptor synaptic vesicle glycoprotein 2 (SV2) was explored in vitro. Their immune response characteristics and protective efficacy were investigated in animal models. The recombinant THc-linker-AHc and AHc-linker-THc proteins with the binding activity had the correct size and structure, thus representing novel subunit vaccines. THc-linker-AHc and AHc-linker-THc induced high levels of specific neutralizing antibodies, and showed strong immune protective efficacy against both toxins. The high antibody titers against the two novel fusion domain molecules and against individual THc and AHc suggested that the THc and AHc domains, as antigens in the fusion functional domain molecules, do not interact with each other and retain their full key epitopes responsible for inducing neutralizing antibodies. Thus, the recombinant THc-linker-AHc and AHc-linker-THc molecules are strong and effective bivalent biotoxin vaccines, protecting against two biotoxins simultaneously. Our experimental design will be valuable to develop recombinant double-RBD fusion molecules as potent bivalent subunit vaccines against bio-toxins. KEY POINTS: • Double-RBD fusion molecules from two toxins had the correct structure and activity. • THc-linker-AHc and AHc-linker-THc efficiently protected against both biotoxins. • Such bivalent biotoxin vaccines based on the RBD are a valuable experimental design.

Biology activity and characterization of the functional L-HN fragment derivative of botulinum neurotoxin serotype E.

OBJECTIVES: The mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. METHODS: Neurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. RESULTS: We found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 µg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. CONCLUSIONS: The EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells.

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (HN and HC ) differ by 13%. The LC acts as a zinc-dependent endopeptidase, HN as the translocation domain, and HC as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, HN of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 HN is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.

Botulinum neurotoxins: new questions arising from structural biology.

Botulinum neurotoxins (BoNTs) are the most toxic substances known and cause botulism in vertebrates. They have also emerged as effective and powerful reagents for cosmetic and medical applications. One important prerequisite for understanding BoNT function in disease, and the further development of the toxins for cosmetic and medical applications, is a detailed knowledge of BoNT interactions with non-toxic neurotoxin-associated proteins and cell surface receptors. Based on the substantial recent progress in obtaining high-resolution crystal structures of key BoNT complexes, we summarize the major advances in understanding BoNT interactions and discuss the resulting potential implications, in particular those relating to BoNT serotype A.

High resolution crystal structures of Clostridium botulinum neurotoxin A3 and A4 binding domains.

Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening condition, botulism. However, while they have the potential to cause serious harm, they are increasingly being utilised for therapeutic applications. BoNTs comprise of seven distinct serotypes termed BoNT/A through BoNT/G, with the most widely characterised being sub-serotype BoNT/A1. Each BoNT consists of three structurally distinct domains, a binding domain (HC), a translocation domain (HN), and a proteolytic domain (LC). The HC domain is responsible for the highly specific targeting of the neurotoxin to neuronal cell membranes. Here, we present two high-resolution structures of the binding domain of subtype BoNT/A3 (HC/A3) and BoNT/A4 (HC/A4) at 1.6 Šand 1.34 Šresolution, respectively. The structures of both proteins share a high degree of similarity to other known BoNT HC domains whilst containing some subtle differences, and are of benefit to research into therapeutic neurotoxins with novel characteristics.

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

Binding behaviors of the HN and the HC domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A HN and HC regions were prepared (HN519-845 and HC967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of HN519-845 to Neuro 2a cells without affecting HC967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both HC967-1296 and HN519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both HC967-1296 and HN519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K+) depletion buffer lowered the binding of both HC967-1296 and HN519-845 to the cells, but seemed to exert a more pronounced effect on the binding of HC967-1296 than on the binding of HN519-845. Results indicate that while both the HN and HC domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells.

Reoccurrence of botulinum neurotoxin subtype A3 inducing food-borne botulism, Slovakia, 2015.

A case of food-borne botulism occurred in Slovakia in 2015. Clostridium botulinum type A was isolated from three nearly empty commercial hummus tubes. The product, which was sold in Slovakia and the Czech Republic, was withdrawn from the market and a warning was issued immediately through the European Commission's Rapid Alert System for Food and Feed (RASFF). Further investigation revealed the presence of botulinum neurotoxin (BoNT) subtype BoNT/A3, a very rare subtype implicated in only one previous outbreak (Loch Maree in Scotland, 1922). It is the most divergent subtype of BoNT/A with 15.4% difference at the amino acid level compared with the prototype BoNT/A1. This makes it more prone to evading immunological and PCR-based detection. It is recommended that testing laboratories are advised that this subtype has been associated with food-borne botulism for the second time since the first outbreak almost 100 years ago, and to validate their immunological or PCR-based methods against this divergent subtype.

Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.

Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus.

Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE: Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid-based expression systems since expressing antigens from a chromosomal location confers an advantage to the vaccine strains by eliminating the antibiotic maintenance required for plasmids and negates issues with plasmid instability that would result in loss of the antigen. Lactic acid bacteria, including Lactobacillus acidophilus, have shown potential for mucosal vaccine delivery, as L. acidophilus is bile and acid tolerant, allowing transit through the gastrointestinal tract where cells interact with host epithelial and immune cells, including dendritic cells. In this study, we successfully expressed C. botulinum and B. anthracis antigens in the probiotic L. acidophilus strain NCFM. Both antigens were highly expressed individually or in tandem from the chromosome of L. acidophilus.

TAT-BoNT/A(1₋448), a novel fusion protein as a therapeutic agent: analysis of transcutaneous delivery and enzyme activity.

Botulinum neurotoxin type A (BoNT/A) has been used as an injectable therapeutic agent for the treatment of some abnormal muscle contractions. In this study, TAT(47-57) peptide, a cell-penetrating peptide, was fused with the catalytic domain of BoNT/A for therapeutic purposes. HeLa and BE(2)-C cell lines were treated separately with purified TAT-BoNT/A(1-448) recombinant protein, and transduction of protein was analyzed by western blotting. Also, transcutaneous delivery through mouse skin surface was evaluated by immunohistochemistry. The in vitro catalytic activity of TAT-BoNT/A(1-448) was evaluated by HPLC. The presence of recombinant protein was detected in both of the cell lines as well as mouse skin cryosections after 60 and 120 min of incubation. The concentration of intracellular proteins was increased over time. HPLC analysis showed that this fusion protein has a biological activity 1.5 times as much as the full-length BoNT/A(1-448) protein. TAT-BoNT/A(1-448) fusion protein is biologically active and can transmit through living cells in vitro and in vivo successfully and more effectively compared with BoNT/A(1-448) protein as control.

Detection of Clostridium botulinum neurotoxin genes (A-F) in dairy farms from Northern Germany using PCR: A case-control study.

Classical botulism in cattle mainly occurs after ingestion of feed contaminated with preformed toxin. In 2001 a form of botulism ("visceral botulism") was postulated to occur after ingestion of Clostridium (C.) botulinum cells or spores, followed by colonization of the intestine, and local production of botulinum neurotoxin (BoNT) causing chronic generalized disease. To verify the potential role of C. botulinum in the described syndrome, a case-control study was conducted, including 139 farms. Fecal samples, rumen content, water and silage samples were collected on each farm. Real time BoNT gene PCR assays were conducted after enrichment in RCM (Reinforced Clostridial Medium) at 37 °C and conventional PCRs after enrichment in MCM (Modified Cooked Meat Medium) at 30 °C. Furthermore, a direct detection of BoNT genes without prior enrichment was attempted. BoNT A, B, C, D, E and F genes were detected in animal samples from 25 (17.99%), 3 (2.16%), 0 (0.0%), 2 (1.44%), 1 (0.72%), and 3 (2.16%) farms, respectively. Eleven feed samples were positive for BoNT A gene. By enrichment a significant increase in sensitivity was achieved. Therefore, this should be an essential part of any protocol. No significant differences regarding BoNT gene occurrence could be observed between Case and Control farms or chronically diseased and clinically healthy animals within the particular category. Thus, the postulated form of chronic botulism in cows could not be confirmed. This study supports the general opinion that C. botulinum can occasionally be found in the rumen and intestine of cows without causing disease.

The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.

Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales, Australia.

Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont/B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST; however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by >3,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST.

First report of an infant botulism case due to Clostridium botulinum type Af.

Most infant botulism cases worldwide are due to botulinum toxin types A and B. Rarely, Clostridium botulinum strains that produce two serotypes (Ab, Ba, and Bf) have also been isolated from infant botulism cases. This is the first reported case of infant botulism due to C. botulinum type Af worldwide.

Identification of fibroblast growth factor receptor 3 (FGFR3) as a protein receptor for botulinum neurotoxin serotype A (BoNT/A).

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502.

Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

Expression, purification and characterization of the receptor-binding domain of botulinum neurotoxin serotype B as a vaccine candidate.

The receptor-binding domain of botulinum neurotoxins (the HC fragment) is a promising vaccine candidate. Among the HC fragments of the seven BoNT serotypes, the expression of HC/B in Escherichia coli is considered especially challenging due to its accumulation as a non-soluble protein aggregate. In this study, the effects of different parameters on the expression of soluble HC/B were evaluated using a screening assay that included growing the bacterium at a small scale, a chemical cell lysis step, and a specific ELISA. The highest soluble HC/B expression levels were obtained when the bacterium E. coli BL21(DE3)+pET-9a-HC/B was grown in terrific broth media at 18°C without induction. Under these conditions, the yield was an order of magnitude higher than previously reported. Standard purification of the protein using a nickel column resulted in a low purity of HC/B. However, the addition of an acidic wash step prior to protein elution released a major protein contaminant and significantly increased the purity level. Mass spectrometry analysis identified the contaminant as ArnA, an E. coli protein that often contaminates recombinant His-tagged protein preparations. The purified HC/B was highly immunogenic, protecting mice from a 10(6) LD50 challenge after a single vaccination and generating a neutralizing titer of 50IU/ml after three immunizations. Moreover, the functionality of the protein was preserved, as it inhibited BoNT/B intoxication in vivo, presumably due to blockade of the neurotoxin protein receptor synaptotagmin.

Targeted delivery of a SNARE protease to sensory neurons using a single chain antibody (scFv) against the extracellular domain of P2X(3) inhibits the release of a pain mediator.

P2X3 (P2X purinoceptor 3) is predominantly expressed on nociceptive sensory neurons and plays a crucial role in signalling leading to chronic inflammatory pain and some features of neuropathic pain. Thus it represents a potential target for pain therapeutics. BoNT/A (botulinum neurooxin type A) effectively relieves certain types of pain through inhibiting the neuronal release of pain peptides. A recombinant single-chain variable fragment (scFv) antibody designated MH7C was generated against the extracellular domain of P2X3 using phage display. The genes encoding the scFv and activated di-chain form of BoNT/A without the C-terminal-binding subdomain (LC-HN-HCN/A) were ligated and expressed in Escherichia coli cells as a composite fusion protein. The purified protein bound and entered P2X3-containing sensory neurons, cleaved synaptosomal-associated protein of 25 kDa and inhibited the release of a pain peptide. This novel fusion protein designated 'LC-HN-HCN/A-MH7C' has potential clinical applications in the treatment of chronic inflammatory and sympathetically maintained neuropathic pain.