Posttranslational Regulation of Botulinum Neurotoxin Production in Clostridium botulinum Hall A-hyper.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind and are the causative agents of the neuroparalytic disease botulism. Despite the overall importance of BoNTs in public health and safety, as a bioterrorism concern, and in pharmaceutical development, little is known about the molecular mechanisms mediating BoNT stability and degradation in various environments. Previous studies using Clostridium botulinum strain ATCC 3502 revealed that high levels of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold. In the present study, the mechanisms of toxin reduction in arginine-enriched cultures of C. botulinum strain Hall A-hyper, which we have previously genetically manipulated using ClosTron technology, were explored. Cultures were grown in toxin production medium (TPM) and TPM enriched with arginine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), changes in pH, and BoNT formation and stability. Our data indicate that arginine enrichment of C. botulinum strain Hall A-hyper cultures results in a pH shift that induces pH-dependent posttranslational control mechanisms. We further show that independent of arginine, maintenance of an acidic culture pH during growth of C. botulinum strain Hall A-hyper plays a central role in toxin stability and that an extracellular metalloprotease produced by the culture results in BoNT degradation at pH levels between ⁓6.5 and 8.0. IMPORTANCE Botulinum neurotoxin (BoNT) is a public health and bioterrorism concern as well as an important and widely used pharmaceutical, yet the regulation of its synthesis by BoNT-producing clostridia is not well understood. This paper highlights the role of environmentally controlled posttranslational regulatory mechanisms influencing processing and stability of biologically active BoNTs produced by C. botulinum. The results of this work will help enhance public health and safety measures and our ability to evaluate safety risks of novel BoNTs and improve production and quality of BoNTs for pharmaceutical use.

Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species.

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria.

The Mechanism of the Beneficial Effect of Botulinum Toxin Type a Used in the Treatment of Temporomandibular Joints Dysfunction.

In the course of temporomandibular joint, dysfunctions very often occur to the excessive increase in tension of masticatory muscles, so the main aim of the treatment is reduction of this hypertension of muscles. For this reason, we use botulinum toxin type A, which is produced by Grampositive Clostridium bacteria. There are six serotypes of the toxin: A, B, C, D, E, F, and G. The botulinum toxin type A was first isolated in 1920s. Today, botulinum toxin type A is used increasingly more often as an efficient and patient-friendly therapy in neurology, ophthalmology, neurology, urology and laryngology. The aim of the article was to review the literature and description of the current knowledge concerned with mechanism of action of botulinum toxin type A, clinical applications and metabolic determinants of muscle contraction and the beneficial effect of this drug on the state of muscle tension.

An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production.

Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics.

Expression and purification of recombinant TAT-BoNT/A(1-448) under denaturing and native conditions.

Botulinum toxin type A can temporarily inhibit muscle contraction. Currently, physicians administer this toxin as a bio-drug in treatment of some muscle contraction disorders. TAT-BoNT/A(1-448) is a functional recombinant protein derived from botulinum toxin light chain. Unlike the full length botulinum toxin, TAT-BoNT/A(1-448) is a self-permeable molecule which can pass through bio-surfaces so can be used as a topical therapeutic agent without injection. To maintain the functionality of TAT-BoNT/A(1-448), it is necessary to restore its normal folding upon expression and purification. In this study, we have investigated and optimized expression conditions for this novel recombinant protein. Under denaturing condition (1 mM IPTG, at 37°C), the chimeric protein was produced as inclusion body and required to be purified using denaturing agents (e.g. urea). Yet, lower incubation temperature (18°C) and less IPTG concentration (0.5 mM) induce a protein under native condition. In such condition, about 60% of the chimeric protein was expressed in soluble form.

Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D.

Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 µg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D.

The pattern of growth observed for Clostridium botulinum type A1 strain ATCC 19397 is influenced by nutritional status and quorum sensing: a modelling perspective.

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to mankind. However, toxin regulation and signals triggering synthesis as well as the regulatory network and actors controlling toxin production are unknown. Experiments show that the neurotoxin gene is growth phase dependent for C. botulinum type A1 strain ATCC 19397, and toxin production is influenced both by culture conditions and nutritional status of the medium. Building mathematical models to describe the genetic and molecular machinery that drives the synthesis and release of BoNT requires a simultaneous description of the growth of the bacterium in culture. Here, we show four plausible modelling options which could be considered when constructing models describing the pattern of growth observed in a botulinum growth medium. Commonly used bacterial growth models are unsuitable to fit the pattern of growth observed, since they only include monotonic growth behaviour. We find that a model that includes both the nutritional status and the ability of the cells to sense their surroundings in a quorum-sensing manner is most successful at explaining the pattern of growth obtained for C. botulinum type A1 strain ATCC 19397.

Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.

RNAi suppression of rice endogenous storage proteins enhances the production of rice-based Botulinum neutrotoxin type A vaccine.

Mucosal vaccines based on rice (MucoRice) offer a highly practical and cost-effective strategy for vaccinating large populations against mucosal infections. However, the limitation of low expression and yield of vaccine antigens with high molecular weight remains to be overcome. Here, we introduced RNAi technology to advance the MucoRice system by co-introducing antisense sequences specific for genes encoding endogenous rice storage proteins to minimize storage protein production and allow more space for the accumulation of vaccine antigen in rice seed. When we used RNAi suppression of a combination of major rice endogenous storage proteins, 13 kDa prolamin and glutelin A in a T-DNA vector, we could highly express a vaccine comprising the 45 kDa C-terminal half of the heavy chain of botulinum type A neurotoxin (BoHc), at an average of 100 µg per seed (MucoRice-BoHc). The MucoRice-Hc was water soluble, and was expressed in the cytoplasm but not in protein body I or II of rice seeds. Thus, our adaptation of the RNAi system improved the yield of a vaccine antigen with a high molecular weight. When the mucosal immunogenicity of the purified MucoRice-BoHc was examined, the vaccine induced protective immunity against a challenge with botulinum type A neurotoxin in mice. These findings demonstrate the efficiency and utility of the advanced MucoRice system as an innovative vaccine production system for generating highly immunogenic mucosal vaccines of high-molecular-weight antigens.

High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli.

The carboxylic domain of the Clostridium botulinum neurotoxin heavy chain (BoNT/A-HC), which has been reported as a vaccine candidate, contains the principle protective antigenic determinants. In this study, the high level expression of the BoNT/A-Hc was achieved by high cell density cultivation of recombinant Escherichia coli in a 2-l batch stirred-tank bioreactor. In order to maximize protein expression, post-induction time and IPTG inducer concentration were optimized by the Taguchi statistical design method. Results showed that the middle of the logarithmic phase and an IPTG concentration of 1 mM presented the optimum conditions for the maximum expression of BoNT/A-HC. High cell density cultivation was subsequently carried out as an effective strategy for the high level expression of recombinant BoNT/A-Hc. Consequently, soluble BoNT/A-Hc was produced at the maximum level of 486 mg l(-1), at 3 h post-induction, which was approximately 9.3 and 7.8 times higher than the levels produced by the shake flask and batch culturing methods, respectively.

New trends in the science of botulinum toxin-A as applied in dystonia.

New trends and advances in botulinum toxin neuroscience paved the way to the better understanding of the toxin from its behavior at the bench to the clinics. As details of mechanism of action are clarified, we are ready to dispose of product myths, such as diffusion being related to the product being used. Our directions, more precisely on the important subjects such as dose targeting, will translate the science to robust clinical information. Additionally, aspects such as antibody formation, leading to treatment nonresponse, are now understood after long-term treatment series in a variety of dystonic conditions. Avenues leading to combinations of these areas of knowledge will lead to improved botulinum neurotoxin (BoNT) treatment, and further explore the potential of this toxin treatment in association with other treatment modalities, particularly in spasticity and dystonia.

Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.

An efficient drug delivery vehicle for botulism countermeasure.

BACKGROUND: Botulinum neurotoxin (BoNT) is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. An effective medical countermeasure strategy would require developing a drug that could rescue poisoned neuromuscular synapses and include its efficient delivery specifically to poisoned presynaptic nerve terminals. Here we report a drug delivery strategy that could directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol. RESULTS: A targeted delivery vehicle was developed for intracellular transport of emerging botulinum neurotoxin antagonists. The drug delivery vehicle consisted of the non-toxic recombinant heavy chain of botulinum neurotoxin-A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted neurons. Our results indicated that the drug delivery vehicle entry into neurons was via BoNT-A receptor mediated endocytosis. Once internalized into neurons, the drug carrier component separated from the drug delivery vehicle in a fashion similar to the separation of the BoNT-A light chain from the holotoxin. This drug delivery vehicle could be used to deliver BoNT-A antidotes into BoNT-A intoxicated cultured mouse spinal cord cells. CONCLUSION: An effective BoNT-based drug delivery vehicle can be used to directly deliver toxin inhibitors into intoxicated nerve terminal cytosol. This approach can potentially be utilized for targeted drug delivery to treat other neuronal and neuromuscular disorders. This report also provides new knowledge of endocytosis and exocytosis as well as of BoNT trafficking.

Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit vaccines against toxin subtypes A1, A2, and A3.

A recombinant, catalytically inactive Clostridium botulinum neurotoxin A1 holoprotein (ciBoNT/A1 HP) was constructed by introducing amino acid substitutions H223A, E224A, and H227A in the active site to ablate proteolytic activity. ciBoNT/A1 HP was produced in the yeast Pichia pastoris and the purified product was evaluated as a vaccine candidate by comparison against recombinant BoNT/A1 LC, LC-belt, LC-H(n), and H(c) antigens and a LC-H(n)+H(c) combination in mouse potency and efficacy bioassays when challenged with BoNT/A subtypes /A1, /A2, and /A3. A single dose of ciBoNT/A1 HP provided equivalent or greater protective immunity, not only against the homologous toxin, but also against two distinct toxin subtypes with significant amino acid divergence. Only the LC-H(n)+H(c) combination provided comparable protection against /A1; however, it was less effective against subtypes /A2 and /A3. Differences in protective immunity diminished after multiple vaccinations with either ciBoNT/A1 HP or BoNT/A1 H(c), and the survival rates were more comparable at the toxin levels used to challenge.

Evaluation of the effect of acetylsalicylic acid on Clostridium botulinum growth and toxin production.

The Republic of Georgia (ROG) has the highest incidence of botulism among all countries in the world, with most cases attributed to home-preserved vegetables. Based on epidemiologic data, the occurrence of botulism in ROG is lower in areas where aspirin (active ingredient, acetylsalicylic acid [ASA]) is added to home-canned vegetables. The objective of this study was to evaluate, with a broth medium, the antibotulinal activity of ASA to determine the possible role of ASA in preventing botulinum toxin production in home-canned vegetables. Trypticase-peptone-glucose-yeast (TPGY) broth (pH 7.0) with 0, 0.3, and 0.6 mg of ASA per ml was inoculated with a 10-strain mixture of proteolytic Clostridium botulinum type A and B spores at ca. 10(3) spores per ml. The inoculated broths were incubated at 31 degrees C under anaerobic conditions, and C. botulinum growth and botulinum toxin production were determined for up to 36 h. Results showed ASA in broth delayed (time to initial detectable toxin produced and amount of toxin produced), but did not prevent, both growth and toxin production by C. botulinum. These results would not provide a definitive explanation for differences in toxin production in canned vegetables prepared with and without aspirin.

High-level expression of the Hcc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen.

A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%-53% of soluble total proteins) and the purified yield was more than 30 mg/L by one-step purification. Then, the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHe protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.

Interaction of syntaxin 1A with the N-terminus of Kv4.2 modulates channel surface expression and gating.

Kv4.2 channels are responsible in the heart for the Ca2+-independent transient outward currents and are important in regulating myocardial excitability and Ca2+ homeostasis. We have identified previously the expression of syntaxin 1A (STX1A) on the cardiac ventricular myocyte plasma membranes, and its modulation of cardiac ATP-sensitive K+ channels. We speculated that STX1A interacts with other cardiac ion channels, thus we examined the interaction of STX1A with Kv4.2 channels. Co-immunoprecipitation and GST pulldown assays demonstrated a direct interaction of STX1A with the Kv4.2 N-terminus. We next investigated the functional alterations of Kv4.2 gating by STX1A in Xenopus oocytes. Coexpression of Kv4.2 with STX1A (1) resulted in a reduction of Kv4.2 current amplitude; (2) caused a depolarizing shift of the steady-state inactivation curve; (3) enhanced the rate of current decay; and (4) accelerated the rate of recovery from inactivation. Additional coexpression of botulinum neurotoxin C, which cleaves STX1A, reversed the effects of STX1A on Kv4.2. STX1A inhibited partially the gating changes by KChIP2, suggesting a competitive interaction of these proteins for an overlapping binding region on the N-terminus of Kv4.2. Indeed, the N-terminal truncation mutants of Kv4.2 (Kv4.2Delta2-40 and Kv4.2Delta7-11), which form part of the KChIP2 binding site, displayed reduced sensitivity to STX1A modulation. Our study suggests that STX1A directly modulates Kv4.2 current amplitude and gating through its interaction with an overlapping region of the KChIP binding motif domain on the Kv4.2 N-terminus.

Variations in expression and release of botulinum neurotoxin in Clostridium botulinum type A strains.

In this study, we characterized the production and release of botulinum neurotoxin during growth of two Clostridium botulinum strains, Hall A and NCTC2916, of distinct gene organizations. The intra- and extracellular fractions of the bacterial cells harvested at various stages of growth were analyzed for the neurotoxin. Both strains exhibited a temporal neurotoxin gene expression; however, these two strains differ in their patterns of growth, toxin production, toxin release, and post-translational nicking. Comparing to the NCTC2916 strain, the Hall A strain showed an extended stationary phase, delayed autolysis, and earlier expression and release of neurotoxin. Understanding the differences between these two toxin-producing strains may provide insights into the toxinogenesis of C. botulinum.

Development and application of Real-Time PCR assays to detect fragments of the Clostridium botulinum types A, B, and E neurotoxin genes for investigation of human foodborne and infant botulism.

Real-time PCR assays for detection of Clostridium botulinum neurotoxin (BoNT) gene fragments specific to BoNTA, B, and E were developed as alternatives to the mouse bioassay. The expected specificities of the PCR assays were demonstrated by in silico analysis as well as empirical testing of target DNA extracted from 83 pure cultures of C. botulinum, and 44 bacteria from other species. The sensitivities of the assays were found to be equivalent to 16, 10, and 141 genomes for BoNT A, B, and E, respectively. The assays were shown to be applicable to both purified DNA, as well as crude DNA extracted from cultures and enrichment broths. The assays were evaluated using DNA extracted directly from clinical and food specimens as well as from inoculated broths using material collected from seven confirmed and one suspected case of botulism. The appropriate BoNT genes were detected in material from seven of the eight cases of botulism and provided a supportive diagnosis faster than the conventional bioassay. These assays have already proven useful for pubic health microbiological investigation of suspected cases of human botulism by substantially improving the diagnostic process.