Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

First confirmed case of infant botulism in Africa, caused by a dual-toxin-producing Clostridium botulinum strain.

Botulism, a rare life-threatening toxemia, is probably underdiagnosed in all of its forms in Africa. This study reports the first laboratory-supported case of infant botulism on the African continent. A 10-week-old, previously well infant presented with progressive global weakness, feeding difficulty, and aspiration pneumonia. During a lengthy hospitalization, a rare bivalent Clostridium botulinum strain, producing subtype B3 and F8 toxins and with a new multilocus sequence type, was isolated from stool. The infant was successfully treated with a heptavalent botulinum antitoxin infusion and pyridostigmine. Despite the relative rarity of infant botulism, this case illustrates the importance of maintaining a high level of clinical suspicion when assessing hypotonic infants. The value of modern diagnostic modalities in identifying and characterizing this under-recognized condition is also demonstrated.

Genomic Characterization of Newly Completed Genomes of Botulinum Neurotoxin-Producing Species from Argentina, Australia, and Africa.

Botulinum neurotoxin-producing clostridia are diverse in the types of toxins they produce as well as in their overall genomic composition. They are globally distributed, with prevalent species and toxin types found within distinct geographic regions, but related strains containing the same toxin types may also be located on distinct continents. The mechanisms behind the spread of these bacteria and the independent movements of their bont genes may be understood through examination of their genetic backgrounds. The generation of 15 complete genomic sequences from bacteria isolated in Argentina, Australia, and Africa allows for a thorough examination of genome features, including overall relationships, bont gene cluster locations and arrangements, and plasmid comparisons, in bacteria isolated from various areas in the southern hemisphere. Insights gained from these examinations provide an understanding of the mechanisms behind the independent movements of these elements among distinct species.

Construction of functional chimeras of syntaxin-1A and its yeast orthologue, and their application to the yeast cell-based assay for botulinum neurotoxin serotype C.

BACKGROUND: Botulinum neurotoxins (BoNTs) prevent synaptic transmission because they hydrolyze synaptic N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). BoNT serotype C (BoNT/C) targets syntaxin-1A and SNAP-25, and is expected to be applied to cosmetic and therapeutic uses. SNAREs are evolutionally conserved proteins and in yeast a syntaxin-1A orthologue Sso1 is involved in exocytosis. The substrate specificity of BoNT/C is strict and it cannot cleave Sso1. METHODS: Domain swapping and mutational screenings were performed to generate functional chimeras SNAREs of syntaxin-1A and Sso1. Such chimeras are expressed in yeast cells and assessed whether they are susceptible to BoNT/C digestion. RESULTS: The Sso1 and syntaxin-1A chimera (Sso1/STX1A), in which the SNARE domain in Sso1 was replaced with that of syntaxin-1A, was not functional in yeast. The functional incompatibility of Sso1/STX1A was attributable to its accumulation in the ER. We found several mutations that could release Sso1/STX1A from the ER to make the chimera functional in yeast. Yeast cells harboring the mutant chimeras grew similarly to wild-type cells. However, unlike wild-type, yeast harboring the mutant chimeras exhibited a severe growth defect upon expression of BoNT/C. Results of further domain swapping analyses suggest that Sso1 is not digested by BoNT/C because it lacks a binding region to BoNT/C (α-exosite-binding region). CONCLUSIONS: We obtained functional Sso1/STX1A chimeras, which can be applied to a yeast cell-based BoNT/C assay. BoNT/C can recognize these chimeras in a similar manner to syntaxin-1A. GENERAL SIGNIFICANCE: The yeast cell-based BoNT/C assay would be useful to characterize and engineer BoNT/C.

Genomics of the Pathogenic Clostridia.

Whole-genome sequences are now available for all the clinically important clostridia and many of the lesser or opportunistically pathogenic clostridia. The complex clade structures of C. difficile, C. perfringens, and the species that produce botulinum toxins have been delineated by whole-genome sequence analysis. The true clostridia of cluster I show relatively low levels of gross genomic rearrangements within species, in contrast to the species of cluster XI, notably C. difficile, which have been found to have very plastic genomes with significant levels of chromosomal rearrangement. Throughout the clostridial phylotypes, a large proportion of the strain diversity is driven by the acquisition and loss of mobile elements, including phages, plasmids, insertion sequences, and transposons. Genomic analysis has been used to investigate the diversity and spread of C. difficile within hospital settings, the zoonotic transfer of isolates, and the emergence, origins, and geographic spread of epidemic ribotypes. In C. perfringens the clades defined by chromosomal sequence analysis show no indications of clustering based on host species or geographical location. Whole-genome sequence analysis helps to define the different survival and pathogenesis strategies that the clostridia use. Some, such as C. botulinum, produce toxins which rapidly act to kill the host, whereas others, such as C. perfringens and C. difficile, produce less lethal toxins which can damage tissue but do not rapidly kill the host. The genomes provide a resource that can be mined to identify potential vaccine antigens and targets for other forms of therapeutic intervention.

C3 Transferase Gene Therapy for Continuous RhoA Inhibition.

The identification of RhoA inhibition as a therapeutic target in neurodegenerative diseases and traumatic central nervous system (CNS) injuries has introduced a need to develop tools that effectively modulate intracellular RhoA-dependent signaling. In neurons, the bacterial exoenzyme C3 transferase irreversibly inactivates RhoA GTPase signaling to promote neuritogenesis and axon regeneration following an injury. Thus, we have adopted a gene therapy approach for the targeted inhibition of RhoA activity in the CNS by expressing C3 transferase. Herein we describe the construction of adeno-associated viral vectors for the expression of cell-permeable-C3 transferase and their functional characterization in vitro.

Inhibition of Clostridium botulinum in Model Reduced-Sodium Pasteurized Prepared Cheese Products.

The 1986 Food Research Institute-Tanaka et al. model predicts the safety of shelf-stable process cheese spread formulations using the parameters of moisture, pH, NaCl, and disodium phosphate (DSP) to inhibit toxin production by Clostridium botulinum. Although this model is very reliable for predicting safety for standard-of-identity spreads, the effects of additional factors have not been considered. The objective of this study was to create a predictive model to include the interactive effect of moisture, pH, fat, sorbic acid, and potassium-based replacements for NaCl and DSP to reflect modern reduced-sodium recipes. Eighty formulations were identified using a central composite design targeting seven factors: 50 to 60% moisture, pH 5.4 to 6.2, 0 to 0.2% sorbic acid, 10 to 30% fat, 1.7 to 2.4% NaCl, 0.8 to 1.6% DSP, and 0 to 50% potassium replacement for sodium salts. Samples were inoculated with proteolytic C. botulinum spores at 3 log spores per g, hot filled into sterile vials, and stored anaerobically at 27°C. Samples were assayed at 0, 1, 2, 3, 4, 8.5, 17.5, 26, and 40 weeks for the presence of botulinum toxin using the mouse bioassay. A parametric survival model was fit to the censored time-to-toxin data. All linear, quadratic, and pairwise effects were considered for model fit. As hypothesized, the effects of pH, sorbate, moisture, DSP, and NaCl were highly significant (P < 0.001). Fat concentration and potassium replacement effects were significant at P < 0.021 and P < 0.057, respectively. The model consistently predicted the safety failure of the toxic samples, but it also predicted failure for some samples that were not toxic. This model is an adjunct to existing models by adding the factors of potassium salts, fat, and sorbic acid to predict the botulinal safety of prepared process cheese products but is not intended to be a substitute for formulation evaluation by a competent process authority.

Neurotoxin synthesis is positively regulated by the sporulation transcription factor Spo0A in Clostridium botulinum type E.

Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here, we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls. Complementation with spo0A restored BoNT production and sporulation. Recombinant DNA-binding domain of Spo0A directly bound to a putative Spo0A-binding box (CTTCGAA) within the BoNT/E operon promoter, demonstrating direct regulation. Spo0A is the first neurotoxin regulator reported in C. botulinum type E. Unlike other C. botulinum strains that are terrestrial and employ the alternative sigma factor BotR in directing BoNT expression, C. botulinum type E strains are adapted to aquatic ecosystems, possess distinct epidemiology and lack BotR. Our results provide fundamental new knowledge on the genetic control of BoNT production and demonstrate common regulation of BoNT production and sporulation, providing a key intervention point for control.

Effect of Equilibrated pH and Indigenous Spoilage Microorganisms on the Inhibition of Proteolytic Clostridium botulinum Toxin Production in Experimental Meals under Temperature Abuse.

Clostridium botulinum is a foreseeable biological hazard in prepared refrigerated meals that needs to be addressed in food safety plans. The objective of this study was to evaluate the effect of product composition and storage temperature on the inhibition of botulinum toxin formation in nine experimental meals (meat, vegetable, or carbohydrate based). Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin in samples stored at 25°C for up to 96 h for phase 1, or at 25°C for 12 h and then transferred to 12.5°C for up to 12 and 6 weeks in phases 1 and 2, respectively. For phase 1, none of the treatments (equilibrated pH 5.8) supported toxin production when stored at 25°C for 48 h, but toxin production was observed in all treatments at 72 h. For the remaining experiments with storage at 12.5°C, toxin production was dependent on equilibrated pH, storage time, and growth of indigenous spoilage microorganisms. In phase 1, no gross spoilage and no botulinum toxin was detected for any treatment (pH ≤5.8) stored at 12.5°C for 12 weeks. In phase 2, gross spoilage varied by commodity, with the brussels sprouts meal with pH 6.5 showing the most rapid spoilage within 2 weeks and botulinum toxin detected at 5 and 6 weeks for the control and cultured celery juice treatments, respectively. In contrast, spoilage microbes decreased the pH of a pH 5.9 beef treatment by 1.0 unit, potentially inhibiting C. botulinum through 6 weeks at 12.5°C. None of the other treatments with pH 5.8 or below supported toxin production or spoilage. This study provides validation for preventive controls in refrigerated meals. These include equilibrated product pH and storage temperature and time to inhibit toxin formation by proteolytic C. botulinum, but the impact of indigenous microflora on safety and interpretation of challenge studies is also highlighted.

Effect of Cultured Celery Juice, Temperature, and Product Composition on the Inhibition of Proteolytic Clostridium botulinum Toxin Production.

Clostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.

Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium.IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical nutrients on growth, lysis, spore formation, BoNT and TC production, and stability of BoNTs of C. botulinum We show that for C. botulinum ATCC 3502 cultured in a complex medium, a high level of arginine repressed BoNT expression by ca. 1,000-fold and also strongly reduced sporulation. Arginine stimulated growth and compensated for a lack of glucose. BoNT and toxin complex proteins were partially inactivated in a complex medium lacking glucose. This work should aid in optimizing BoNT production for pharmaceutical uses, and furthermore, an understanding of the nutritional regulation of growth and BoNT formation may provide insights into growth and BoNT formation in foods and clinical samples and into the enigmatic function of BoNTs in nature.

Effects of transgenic expression of botulinum toxins in Drosophila.

Clostridial neurotoxins (botulinum toxins and tetanus toxin) disrupt neurotransmitter release by cleaving neuronal SNARE proteins. We generated transgenic flies allowing for conditional expression of different botulinum toxins and evaluated their potential as tools for the analysis of synaptic and neuronal network function in Drosophila melanogaster by applying biochemical assays and behavioral analysis. On the biochemical level, cleavage assays in cultured Drosophila S2 cells were performed and the cleavage efficiency was assessed via western blot analysis. We found that each botulinum toxin cleaves its Drosophila SNARE substrate but with variable efficiency. To investigate the cleavage efficiency in vivo, we examined lethality, larval peristaltic movements and vision dependent motion behavior of adult Drosophila after tissue-specific conditional botulinum toxin expression. Our results show that botulinum toxin type B and botulinum toxin type C represent effective alternatives to established transgenic effectors, i.e. tetanus toxin, interfering with neuronal and non-neuronal cell function in Drosophila and constitute valuable tools for the analysis of synaptic and network function.

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.

Recombinant Botulinum Toxoids: A Practical Guide for Production.

Clostridium botulinum is a Gram-positive, spore-forming, anaerobic bacillus that produces a potent neurotoxin. Botulinum neurotoxins (BoNTs) are classified from serotypes A to H, and even though they have similar mechanisms of action, they show preferential hosts. In veterinary medicine, BoNT serotypes C and D are the most important, once several animal species are susceptible to them. Since BoNTs are the most potent toxins known in nature, the best way to control botulism in animals is through vaccination. However, current commercial vaccines are based on inactivated toxins (toxoids) and cells (bacterins) and present many drawbacks, such as a time-consuming production with variable antigen yield and biosafety risks. Recombinant vaccines, especially those produced by Escherichia coli expression system, have proved to be an interesting alternative to overcome these problems. E. coli is a very well-known microorganism that allows the production of large amounts of nontoxic recombinant antigens in a short period using simple culture medium reducing the production complexity and decreasing most of the biosafety risks involved in the process. We describe herein a method for the production of recombinant vaccines for veterinary medicine application, involving initial steps of gene design up to vaccine formulation and evaluation itself.

Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells.

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells.

Characterizing the fecal microbiota of infants with botulism.

BACKGROUND: Infant botulism is the most prevalent form of botulism in the USA, representing 68.5 % of cases reported from 2001-2012. Infant botulism results when botulinum toxin-producing clostridia (BTPC) colonize the infant gut with concomitant in vivo production of the highly potent botulinum neurotoxin (BoNT). The gut microbiota of infants with botulism is largely uncharacterized; therefore, it remains unclear whether the microbiota profile of these patients are distinct in composition, abundance, or diversity. To address this uncertainty, we employed 16S rRNA gene profiling to characterize the fecal microbiota in 14 stool samples among laboratory-confirmed and non-confirmed infant botulism cases. RESULTS: Seven bacterial phyla were identified among all 14 infant stool samples examined. Compared to samples from non-confirmed cases, the fecal microbiota of infant botulism patients displayed significantly higher Proteobacteria abundance. Of the 20 bacterial families identified, Enterobacteriaceae was significantly more abundant in samples from infants with botulism. Firmicutes abundance and the abundance ratio of Firmicutes/Proteobacteria was significantly lower in samples from infants with botulism. Lactobacillus spp. abundance was notably reduced in 12 of the 14 samples. Clostridium botulinum and Clostridium baratii were identified in low relative abundances in confirmed and non-confirmed samples based on their 16S rRNA gene profiles, although their toxigenicity remained undetermined. No significant differences were observed in the number of operational taxonomic units (OTUs) observed or in fecal microbiota diversity between laboratory-confirmed and non-confirmed samples. Correlations between individual phylum abundances and infant age were variable, and no significant differences were shown in number of OTUs observed or in fecal microbiota diversity between samples delineated by overall mean age. CONCLUSIONS: Significant differences in Proteobacteria, Firmicutes, and Enterobacteriaceae abundances were identified in the fecal microbiota of infants with botulism when compared to samples from non-confirmed cases. Fecal microbiota diversity was not significantly altered in infants with botulism, and a limited presence of BTPC was shown. It could not be determined whether the fecal microbiota profiles shown here were comparable prior to patient illness, or whether they were the direct result of infant botulism. The results of this study do, however, provide a detailed and descriptive observation into the infant gut microbiota after intestinal colonization by BTPC.

Clostridium botulinum Toxin Production in Relation to Spoilage of Atlantic Salmon (Salmo salar) Packaged in Films of Varying Oxygen Permeabilities and with Different Atmospheres.

Shelf life of fish packaged under modified atmosphere (MA) is extended, but within the United States, commercial application of MA with impermeable packaging films is restricted due to concerns that botulinum toxin production would precede spoilage when contaminated fish are held at abusive storage temperatures. Use of semipermeable packaging films has been advocated; however, previous studies are inconclusive in determining the oxygen transmission rate (OTR) of a film that is needed to achieve an acceptable margin of safety (i.e., toxin production occurs only after spoilage). This study was conducted to determine the influence of OTR (target OTRs of 3 to 15,000) on the development of spoilage volatiles and toxin in salmon inoculated with type E Clostridium botulinum and subjected to air, vacuum, or 75:25 CO2:N2 MA and storage temperatures of 4, 8, 12, or 16°C. The most dominant headspace volatile peak that was produced during spoilage of samples at 4, 8 or 12°C was a peak, having a Kovats retention index (KI) of 753, and at which external standards of 2- or 3-methyl 1-butanol also eluted. Under anaerobic conditions, both the aerobic microbial populations and the size of the KI 753 spoilage peak were less in inoculated samples compared with uninoculated samples. C. botulinum-inoculated samples that were stored at 12 or 16°C under conditions favorable for anaerobic growth were also characterized by a KI 688 peak. Using a previously developed model that related the percentage of elderly consumers who would prepare a sample having the KI 753 spoilage peak of a specific size, it was determined that for salmon packaged with 3 or 3,000 OTR films under any atmosphere and stored at 12 or 16°C, 2 to 61% of the consumers could potentially prepare toxin-contaminated samples. Hence, when abusive storage conditions are suspected, the fish should not be consumed.

Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme.

C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells.

Expression and biochemical characterization of light chains of Botulinum neurotoxin subtypes F5 and F7.

Botulinum neurotoxins are the most potent protein toxins known to human. To date, seven subtypes of the BoNT/F serotype (BoNT/F1 to BoNT/F7) have been identified, among which BoNT/F5 and BoNT/F7 are the most divergent. However, little structural and functional information is available for these two subtypes due to a lack of suitable recombinant proteins for biochemical characterization, except that they appear to possess unique substrate recognition mechanisms, thereby impeding development of vaccine or inhibitors against these proteins. In the present study, we utilized a combinatorial approach which involved examining the effects of different affinity tags, mapping C-terminal truncation mutants and optimization of expression and purification conditions, that allowed us to successfully express and purify soluble and highly active recombinant LC/F5 and LC/F7 proteins. GST-LC/F5(1-450) and 6× His-LC/F5(1-405) were the formats which exhibit the highest level of solubility and activity, whereas GST-LC/F7(1-405) was the most active form of LC/F7. In comparison, GST-LC/F5(1-450) was more active than GST-LC/F7(1-405), which was in turn more active than the LC/F1 control. Our data suggest that solubility of these proteins strongly correlated with their catalytic activity. Successful expression and purification of LC/F5 and LC/F7 in this work will, for the first time, provide materials for further characterization of these two subtypes of BoNT/F, which is essential for future development of protective vaccine or other therapeutic strategies, as well as BoNT/F protein engineering.

Two-component systems and toxinogenesis regulation in Clostridium botulinum.

Botulinum neurotoxins (BoNTs) are the most potent toxins ever known. They are mostly produced by Clostridium botulinum but also by other clostridia. BoNTs associate with non-toxic proteins (ANTPs) to form complexes of various sizes. Toxin production is highly regulated through complex networks of regulatory systems involving an alternative sigma factor, BotR, and at least 6 recently described two-component systems (TCSs). TCSs allow bacteria to sense environmental changes and to respond to various stimuli by regulating the expression of specific genes at a transcriptional level. Several environmental stimuli have been identified to positively or negatively regulate toxin synthesis; however, the link between environmental stimuli and TCSs is still elusive. This review aims to highlight the role of TCSs as a central point in the regulation of toxin production in C. botulinum.