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Clostridium botulinum is Gram-positive anaerobic spore-forming bacterium that produces very potent neurotoxins that have a potential to be used as bioweapon. In this study, we utilized applications of pan genome to develop drug targets for 51 strains of Clostridium botulinum. From the total 8756 core proteins, there were 422 non-host homologous proteins from which we selected 7 essential proteins by applying 2 thresholds that are identity > 35 and e-value = 0.001. These 7 proteins were selected as drug targets and docked against 105 anti-bacterial compounds. After docking, against each protein, top 10 compounds were selected based on the binding affinity and number of interactions and from these 10 compounds, the most promising compound was ranked on top for all target proteins. Furthermore, we also identified some compounds that have shown highest binding energies with more than one protein. From the 105 anti-bacterial compounds, we got 39 compounds on the basis of their binding energies with all target proteins. These compounds can be further validated by in vitro analysis and can proceed for clinical trials.

Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an effective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (HC/A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant HC/A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand-receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rHC/A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rHC/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 ≈ 27 nM) > FGFR2b (EC50 ≈ 70 nM) > FGFR1c (EC50 ≈ 163 nM); rHC/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 ≈ 18 nM). Mutating the ganglioside binding site in HC/A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein HC/A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization.

Nitrite is added to meat products as a preservative and it acts as a bacteriostatic compound against Clostridium botulinum growth. Nitric-oxide (˙NO), myoglobin and S-nitroso-compounds seem to be the main molecules generated from nitrite in meat products, which by decomposition to ˙NO, form the main anti-clostridial factor. The growth of C. sporogenes from activated spores in the presence of 0.5–2.5 mM NAC–SNO was compared to nitrite, both at 37 °C for 5 days and at room temperature for 28 days. The present study demonstrates that NAC–SNO under the same conditions and concentrations, in meat products, acts as an anti-clostridial compound similar to nitrite. In contrast to nitrite which must be activated in meat by heating, NAC–SNO generates the anti-clostridial factor directly, without heating, as was evaluated in an unheated bacteriological medium. The toxic effect of NAC–SNO and nitrite in methaemoglobinaemia and generation of N-nitrosamines in vivo, in mice, were also determined. Mice were gavage fed milk containing 45 mg per kg per bw of nitrite or an equimolar equivalent of NAC–SNO in the presence of 50 mg per kg per bw of N-methylaniline. Nitrite generated methaemoglobinaemia and carcinogenic N-nitrosoamines (N-nitrosomethylaniline); however, NAC–SNO under the same conditions and concentrations generates much less methaemoglobin and no detectable N-nitrosoamines in the blood, in vivo.

Binary enterotoxins Clostridioides difficile CDT toxin, Clostridium botulinum C2 toxin, and Clostridium perfringens iota toxin consist of two separate protein components. The B-components facilitate receptor-mediated uptake into mammalian cells and form pores into endosomal membranes through which the enzymatic active A-components translocate into the cytosol. Here, the A-components ADP-ribosylate G-actin which leads to F-actin depolymerization followed by rounding of cells which causes clinical symptoms. The protein folding helper enzymes Hsp90, Hsp70, and peptidyl-prolyl cis/trans isomerases of the cyclophilin (Cyp) and FK506 binding protein (FKBP) families are required for translocation of A-components of CDT, C2, and iota toxins from endosomes to the cytosol. Here, we demonstrated that simultaneous inhibition of these folding helpers by specific pharmacological inhibitors protects mammalian, including human, cells from intoxication with CDT, C2, and iota toxins, and that the inhibitor combination displayed an enhanced effect compared to application of the individual inhibitors. Moreover, combination of inhibitors allowed a concentration reduction of the individual compounds as well as decreasing of the incubation time with inhibitors to achieve a protective effect. These results potentially have implications for possible future therapeutic applications to relieve clinical symptoms caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs for their membrane translocation into the cytosol of target cells.

Botulinum neurotoxins (BoNTs) cause the deadly condition called botulism, but they can be used as therapeutics for a wide range of indications. BoNTs are classified into many different serotypes and subtypes, each with potentially different intoxication properties. Here, we report crystal structures of the receptor‐binding domains from two subtypes (BoNT/A5 and BoNT/A6) and compare their binding sites with previous BoNT/A structures., Clostridium botulinum neurotoxins (BoNTs) cause flaccid paralysis through inhibition of acetylcholine release from motor neurons; however, at tiny doses, this property is exploited for use as a therapeutic. Each member of the BoNT family of proteins consists of three distinct domains: a binding domain that targets neuronal cell membranes (HC), a translocation domain (HN) and a catalytic domain (LC). Here, we present high‐resolution crystal structures of the binding domains of BoNT subtypes/A5 (HC/A5) and/A6 (HC/A6). These structures show that the core fold identified in other subtypes is maintained, but with subtle differences at the expected receptor‐binding sites.

A feeding experiment was carried out with late-lactating cows over 12 weeks to evaluate the feeding value of a basic diet with maize and grass silage (MS, GS) when combined with varying portions of concentrate in the ration (20% and 60% on a dry matter basis) and to test the effects on health and performance, the transfer of important Fusarium toxins to blood and milk, the total and Shiga toxin (stx)-forming E. coli counts, and the presence of Clostridium botulinum neurotoxin (BoNT) genes in rectal fecal samples. MS was contaminated by a broader spectrum of fungal and other metabolites compared to GS. MS contained higher concentrations of the important Fusarium toxins deoxynivalenol (DON) and zearalenone (ZEN). Blood and milk levels of DON and ZEN residues generally reflected the differences in exposure at a low level. Feeding of MS with 60% concentrate feed induced subacute ruminal acidosis (SARA) associated with a marked drop in dry matter intake, fat corrected milk yield and a fat to protein ratio in milk of lower than 1. The SARA-associated higher ruminal LPS concentration did not affect the circulating concentrations of haptoglobin as an indicator of systemic inflammation. Lower rumen pH values in both MS-fed groups were associated with lower pH values, higher absolute E. coli counts and increased proportions of stx-positive E. coli in rectal feces. BoNT genes A, B, C, D, E and F remained undetectable in any of the fecal samples suggesting that feedstuffs were virtually free of the corresponding C. botulinum strains. In conclusion, maize feedstuff (silage, grains, starch-containing byproducts)-dominated rations for dairy cows should be avoided to reduce adverse effects on health and food safety.

Botulism is a severe neuroparalytic illness, which affects the nervous system. It is caused by botulinum neurotoxins (BoNTs), produced by anaerobic gram-positive bacteria Clostridium botulinum. There are 7 serotypes of BoNT A-G, but BoNT A/B/D/E plays a major role in botulism affecting humans. Foodborne botulism (classic botulism) is the most frequent clinical manifestation occurring after consumption of food containing botulinum neurotoxins. The diagnosis of botulism is based on clinical symptoms; however, recommended and alternative laboratory methods are used to confirm the etiology of symptoms and the identification of BoNT toxin type. The aim of this work was to present the epidemiology of foodborne botulism in Poland and to gather and analyze the available diagnostic methods that allow us to detect BoNT in clinical samples. Using the epidemiological reports of National Institute of Hygiene in Poland and findings presented in the Przegląd Epidemiologiczny, the incidence of classical botulism in Poland has been presented over a period of recent 18 years. Searching for the optimal diagnostic method for BoNT identification in various samples, we have confronted the sensitivity and specificity of recently available alternative methods with classical biological assay.

Background: Considering the high fatality of botulism, the control of Clostridium botulinum and its neurotoxins has clinical importance. In this regard, using chemical preservatives, natural essential oils (Eos), and changes in the growth predisposing factors of bacteria are suitable methods to control the growth and toxin producing of C. botulinum in foods. Objective: The current survey was done to assess the effects of Citrus sinensis EO and intrinsic and extrinsic factors on the growth and toxin producing of C. botulinum type A. Materials and Methods: In this experiment with a factorial design, C. sinensis EO (0.0%, 0.015%, 0.03%, and 0.045%), nisin (0, 500, and 1500 IU/mL), nitrite (0, 20, and 60 ppm), pH (5.5 and 6.5), storage temperature (25 and 35° C), and sodium chloride (NaCl, 0.5% and 3%) were used to assess bacterial growth in the brain heart infusion medium. Finally, the mouse bioassay method was also used to assess toxicity. Results: Clostridium sinensis EO with a concentration of 0.045%, as well as the reduction of pH and temperature could significantly delay the growth of bacteria (P≤0.05) in contrast to the use of NaCl and nisin alone. However, all concentrations of sodium chloride (NaCl), nisin, and C. sinensis EO (< 0.045%) in interaction with each other, especially in combination with nitrite, showed good synergistic effects. Conclusion: These results suggested that using certain concentrations of C. sinensis EO and nisin, along with other suboptimal factors caused a significant decrease in the nitrite contents of foods with a significant reduction in the growth and toxin-producing ability of C. botulinum.

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

Lectins are proteins with the ability to recognize and bind to specific glycan structures. These molecules play important roles in many biological systems and are actively being studied because of their ability to detect glycan biomarkers for many diseases. Hemagglutinin (HA) proteins from Clostridium botulinum type C neurotoxin complex; HA1, HA2, and HA3 are lectins that aid in the internalization of the toxin complex by binding to glycoproteins on the cell surface. HA1 mutants have been previously reported, namely HA1 W176A/D271F and HA1 N278A/Q279A which are specific to galactose (Gal)/N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) sugars, respectively. In this study, we utilized HA1 mutants and expressed them in complex with HA2 WT and HA3 WT to produce glycan detecting tools with high binding affinity. Particularly, two types were made: Gg and Rn. Gg is an Alexa 488 conjugated lectin complex specific to Gal and GalNAc, while Rn is an Alexa 594 conjugated lectin complex specific to Neu5Ac. The specificities of these lectins were identified using a glycan microarray followed by competitive sugar inhibition experiments on cells. In addition, we confirmed that Gg and Rn staining is clearly different depending on cell type, and the staining pattern of these lectins reflects the glycans present on the cell surface as shown in enzyme treatment experiments. The availability of Gg and Rn provide us with new promising tools to study Gal, GalNAc, and Neu5Ac terminal epitopes which can aid in understanding the functional role of glycans in physiological and pathological events.

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

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.

International journal of molecular sciences 22(15), 8315 (1-12) (2021). doi:10.3390/ijms22158315, The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228–1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides., Published by Molecular Diversity Preservation International, Basel

Cooking temperature of poultry meat is typically inadequate to inactivate the heat resistant spores of Clostridium botulinum. The purpose of this study is to develop a predictive model for C. botulinum during cooling of cooked ground chicken. Cooked chicken was inoculated with a cocktail of five strains of proteolytic C. botulinum type A and five strains of proteolytic C. botulinum type B to yield a final spore concentration of approximately 2 log CFU/g. The growth of C. botulinum was determined at constant temperatures from 10 to 46 °C. Dynamic temperature experiments were performed with continued cooling from 54.4 to 4.4 °C or 7.2 °C in mono- or bi-phasic cooling profiles, respectively. The Baranyi primary model was used to fit growth data and the modified Ratkowsky secondary model was used to fit growth rates with respect to temperature. The primary models fitted the growth data well (R2 values ranging from 0.811 to 0.988). The R2 and root mean square error (RMSE) of the modified Ratkowsky secondary model were 0.95 and 0.06, respectively. Out of 11 prediction error values calculated in this study, ten were within the limit of acceptable prediction zone (−1.0 to 0.5), indicating a good fit of the model. The predictive model will assist institutional food service operations in determining the safety of cooked ground chicken subjected to different cooling periods.

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum and related species cause botulism, a neuroparalytic disease associated with a high mortality. BoNTs are always produced as large protein complexes (progenitor toxin complexes, PTCs) through association with non-toxic components (NAPs) including hemagglutinin (HA) and non-toxic non-hemagglutinin (NTNHA). Food-borne botulism is caused by the ingestion of PTCs. PTCs in the gastrointestinal tract cross the intestinal epithelial barrier, enter the blood stream, and reach the nerve endings, where BoNTs cleave the SNAREs required for vesicle fusion. Consequently, BoNTs inhibit neurotransmitter release and cause paralysis. To cause food-borne botulism, BoNTs must traverse the intestinal epithelial barrier. However, the mechanism used to cross this barrier remains unclear. Using an in vitro epithelial barrier system, we previously showed that the interaction of HA with E-cadherin results in disruption of tight junctions. Furthermore, we previously reported that microfold (M) cells in the follicle-associated epithelium (FAE) of mouse Peyer's patches (PPs) are major sites where type A1 BoNT breaches the intestinal epithelial barrier. Here, I would like to demonstrate an ingenious invasion mechanism of the BoNT complex.

Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.

Clostridium sporogenes has been widely used as a surrogate for proteolytic C. botulinum for validating thermal processes in low-acid cans. To limit the intensity of heat treatments, industrials must use other ways of control as an association of acidic and saline environment after a low heat treatment. The probability of growth of pH (7–4.4), sodium chloride concentration (0–11%) and heat treatment (80°C-10 min; 100°C-1.5 min and 5.2 min) were studied on C. sporogenes PA 3679 spores and vegetative cells. Vegetative cells or heat-treated spores were inoculated in PYGm broth at 30 °C for 48 days in anaerobic conditions. Vegetative cells growth (pH 4.6–pH 4.5; 7%–8% NaCl) range is larger than the spore one (pH 5.2–pH 5.0; 6%–7% NaCl). Spores germination and outgrowth rage is decreased if the spores are heat-treated at 100 °C for 1.5 min (pH 5.5–5.3; 4%–5% NaCl) and 5.2 min (pH 5.7–5.3; 4%–5% NaCl). The C. sporogenes PA 3679 spores germination and outgrowth is impacted by their physiological state. The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) opening new possibilities for optimizing food formulation processes to manage the risks of C. sporogenes spoilage.

Botulinum neurotoxins (BoNTs) produced by soil bacterium Clostridium botulinum are cause of botulism and listed as biohazard agents, thus rapid screening assays are needed for taking the correct countermeasures in a timely fashion. The gold standard method relies on the mouse lethality assay with a lengthy analysis time, i.e., 2–5 days, hindering the prompt management of food safety and medical diagnosis. Herein, we propose the first paper-based antibody-free sensor for reliable and rapid detection of BoNT/A and BoNT/C, exploiting their cleavage capability toward a synthetic peptide able to mimic the natural substrate SNAP-25. The peptide is labelled with the electroactive molecule methylene blue and immobilized on the paper-based electrode modified with gold nanoparticles. Because BoNT/A and BoNT/C can cleave the peptide with the removal of methylene blue from electrode surface, the presence of these neurotoxins in the sample leads to a signal decrease proportional to BoNT amount. The biosensor developed with the selected peptide and combined with smartphone assisted potentiostat is able to detect both BoNT/A and BoNT/C with a linearity up to 1 nM and a detection limit equal to 10 pM. The applicability of this biosensor was evaluated with spiked samples of orange juice, obtaining recovery values equal to 104 ± 6% and 98 ± 9% for 1 nM and 0.5 nM of BoNT/A, respectively.

Synaptic transmission is mediated by the regulated exocytosis of synaptic vesicles. When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the influx of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recycled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is unclear which of these changes are directly linked to SV cycling and which might regulate other presynaptic functions that are also controlled by calcium-dependent kinases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization-induced calcium influx remained unchanged. BoNT-treatment significantly alters the response of the synaptic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca2+-dependent phosphorylation from SV-cycling-dependent phosphorylation and confirm an effect of such SV-cycling-dependent phosphorylation events on syntaxin-1a-T21/T23, synaptobrevin-S75, and cannabinoid receptor-1-S314/T322 on exo- and endocytosis in cultured hippocampal neurons., Graphical Abstract, Highlights • KCl-depolarization induces phosphoproteome changes in isolated nerve terminals (synaptosomes). • Botulinum neurotoxin affects protein phosphorylation in depolarized synaptosomes. • BoNT treatment reveals phosphorylation sites that depend on SV-cycling activity. • SV-cycling-dependent sites on Vamp2, Stx1a, and Cnr1 affect exo- and endocytosis., In Brief Analysis of protein phosphorylation in isolated nerve terminals (synaptosomes) treated with C. botulinum neurotoxins (BoNT) to inhibit synaptic vesicle (SV) cycling reveals phosphorylation events that are primarily dependent on depolarization-induced Ca2+ influx and those that also require active SV-cycling machinery. In particular, SV-cycling-dependent phosphorylation sites on synaptobrevin (Vamp2), syntaxin-1 (Stx1a), and cannabinoid receptor-1 (Cnr1) are capable of changing the rate of exo- and endocytosis in cultured hippocampal neurons.

Fermented meat products are important not only for their sensory characteristics, nutrient content and cultural heritage, but also for their stability and convenience. The aroma of fermented meat products is unique and its formation mechanisms are not completely understood; however, the presence of nitrite and nitrate is essential for the development of cured aroma. The use of nitrite and nitrate as curing agents in meat products is based on its preservation activity. Even though their presence has been associated with several risks due to the formation of nitrosamines, their use is guarantee due to their antimicrobial action against Clostridium botulinum. Recent trends and recommendations by international associations are directed to use nitrite but at the minimum concentration necessary to provide the antimicrobial activity against Clostridium botulinum. This chapter discuss the actual limits of nitrite and nitrite content and their role as curing agents in meat products with special impact on dry fermented products. Regulatory considerations, antimicrobial mechanisms and actual trends regarding nitrite reduction and its effect on sensory and aroma properties are also considered.

Clostridium botulinum poses a serious threat to food safety and public health by producing potent neurotoxin during its vegetative growth and causing life-threatening neuroparalysis, botulism. While high temperature can be utilized to eliminate C. botulinum spores and the neurotoxin, non-thermal elimination of newly germinated C. botulinum cells before onset of toxin production could provide an alternative or additional factor controlling the risk of botulism in some applications. Here we introduce a putative phage lysin that specifically lyses vegetative C. botulinum Group I cells. This lysin, called CBO1751, efficiently kills cells of C. botulinum Group I strains at the concentration of 5 µM, but shows little or no lytic activity against C. botulinum Group II or III or other Firmicutes strains. CBO1751 is active at pH from 6.5 to 10.5. The lytic activity of CBO1751 is tolerant to NaCl (200 mM), but highly susceptible to divalent cations Ca2+ and Mg2+ (50 mM). CBO1751 readily and effectively eliminates C. botulinum during spore germination, an early stage preceding vegetative growth and neurotoxin production. This is the first report of an antimicrobial lysin against C. botulinum, presenting high potential for developing a novel antibotulinal agent for non-thermal applications in food and agricultural industries.

Botulinum neurotoxin (BoNT) is a neurotoxin produced by Clostridium botulinum in an anaerobic environment. BoNT is the most toxic protein among bacteria, animals, plants, and chemical substances reported to date. BoNTs are 150 kDa proteins composed of three major functional domains: catalytic (L domain, 50 kDa), translocation (HN domain, 50 kDa), and receptor-binding (Hc domain, 50 kDa) domains. Most studies have focused on the use of the Hc domain as an antigen because it is capable of generating robust protective immunity and contains some functional neutralizing epitopes. In the present study, we produced and characterized a recombinant L-HN fusion fragment of the parent BoNT/B (BL-HN) composed of L and HN domains with a deletion in the Hc domain (BHc). When the BL-HN protein was expressed in E. coli, it retained its stable structure and antigenicity. As a vaccine antigen, the recombinant BL-HN protein was found to induce sufficient protection against native BoNT/B in a mouse model. The BL-HN subunit vaccine could also induce a strong humoral immune response and generate sufficient neutralizing antibodies in immunized mice. Therefore, BL-HN may retain the native neurotoxin structure and critical epitopes responsible for inducing serum neutralizing antibodies. Studies of the dose-dependent immunoprotective effects further confirmed that the BL-HN antigen could provide potent protective immunity. This finding suggests that BL-HN can play an important role in immune protection against BoNT/B. Therefore, the BL-HN fusion fragment provides an excellent platform for the design of recombinant botulinum vaccines and neutralizing antibodies.

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A’s substrate and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A infiltrates neurons and retains its SNAP23 activity. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.One Sentence SummaryDirected evolution of the BoNT/A protease targets a new cellular protein, SNAP23, expanding its therapeutic potential.

Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. Distantly related species have similar spore architectures with protective proteinaceous layers aiding adhesion and targeting. The structures that confer these common properties are largely unstudied, and the proteins involved can be very dissimilar in sequence. We identify CsxA as a cysteine-rich protein that self-assembles in a two-dimensional lattice enveloping the spores of several Clostridium species. We show that apparently unrelated cysteine-rich proteins from very different species can self-assemble to form remarkably similar and robust structures. We propose that diverse cysteine-rich proteins identified in the genomes of a broad range of spore formers may adopt a similar strategy for assembly., Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives can have similar spore architectures although some display unique features; they all incorporate protective proteinaceous envelopes. We previously found that Bacillus spores can achieve these protective properties through extensive disulfide cross-linking of self-assembled arrays of cysteine-rich proteins. We predicted that this could be a mechanism employed by spore formers in general, even those from other genera. Here, we tested this by revealing in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other clostridial relatives, forms a hexagonally symmetric semipermeable array. A cysteine-rich protein, CsxA, when expressed in Escherichia coli, self-assembles into a highly thermally stable structure identical to that of the native exosporium. Like the exosporium, CsxA arrays require harsh “reducing” conditions for disassembly. We conclude that in vivo, CsxA self-organizes into a highly resilient, disulfide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar to that in Bacillus spores, despite a lack of protein homology. In both cases, intracellular disulfide formation is favored by the high lattice symmetry. We have identified cysteine-rich proteins in many distantly related spore formers and propose that they may adopt a similar strategy for intracellular assembly of robust protective structures. IMPORTANCE Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. Distantly related species have similar spore architectures with protective proteinaceous layers aiding adhesion and targeting. The structures that confer these common properties are largely unstudied, and the proteins involved can be very dissimilar in sequence. We identify CsxA as a cysteine-rich protein that self-assembles in a two-dimensional lattice enveloping the spores of several Clostridium species. We show that apparently unrelated cysteine-rich proteins from very different species can self-assemble to form remarkably similar and robust structures. We propose that diverse cysteine-rich proteins identified in the genomes of a broad range of spore formers may adopt a similar strategy for assembly.

Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease ‘botulism’ in human and animals. Their potential use as biological weapon led to their classification in category ‘A’ biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni–NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4+ and CD8+ T cells. Increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4+ and CD8+ T cells, respectively; in immunized group (P + T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E.

Nitrate and nitrite are the most applied food additives to inhibit microbial growth. The main risk of nitrate and nitrite presence in food is the formation of nitrosamines during food preparation and digestion. However, dietary intake of nitrite and nitrate from food has potential cardiovascular benefits. Accordingly, 160 samples from meat and poultry were randomly collected to evaluate the level of occurrence nitrate and nitrite using an HPLC method and estimate the daily intake (EDI) of Egyptian adults and children. The levels of nitrate and nitrite ranged between 1.3-557 mg/kg and 6-812 mg/kg, respectively. The EDI was compared with the accepted daily intake (ADI) represented in the hazard index (HI) scale. The nitrite results clarify that all HI values were above 1 for all commodities, while for nitrate it exceeded 1 only for canned meat and basturma, when consumed by children.

The botulinum neurotoxin (BoNT) is the most lethal protein known to man causing the deadly disease botulinum. The neurotoxin, composed of a heavy (HC) and light (LC) chain, work in concert to cause muscle paralysis. A therapeutic strategy to treat individuals infected with the neurotoxin is inhibiting the catalytic activity of the BoNT LC. We report the synthesis, inhibition study and computational docking analysis of novel small molecule BoNT/A LC inhibitors. A structure activity relationship study resulted in the discovery of d -isoleucine functionalized with a hydroxamic acid on the C-terminal and a biphenyl with chlorine at C- 2 connected by a sulfonamide linker at the N-terminus. This compound has a measured IC50 of 0.587 µM for the BoNT/A LC. Computational docking analysis indicates the sulfonamide linker adopts a geometry that is advantageous for binding to the BoNT LC active site. In addition, Arg363 is predicted to be involved in key binding interactions with the scaffold in this study.

Clostridium tetani produces a potent neurotoxin, the tetanus toxin (TeNT), which is responsible for an often-fatal neurological disease (tetanus) characterized by spastic paralysis. Prevention is efficiently acquired by vaccination with the TeNT toxoid, which is obtained by C. tetani fermentation and subsequent purification and chemical inactivation. C. tetani synthesizes TeNT in a regulated manner. Indeed, the TeNT gene (tent) is mainly expressed in the late exponential and early stationary growth phases. The gene tetR (tetanus regulatory gene), located immediately upstream of tent, encodes an alternative sigma factor which was previously identified as a positive regulator of tent. In addition, the genome of C. tetani encodes more than 127 putative regulators, including 30 two-component systems (TCSs). Here, we investigated the impact of 12 regulators on TeNT synthesis which were selected based on their homology with related regulatory elements involved in toxin production in other clostridial species. Among nine TCSs tested, three of them impact TeNT production, including two positive regulators that indirectly stimulate tent and tetR transcription. One negative regulator was identified that interacts with both tent and tetR promoters. Two other TCSs showed a moderate effect: one binds to the tent promoter and weakly increases the extracellular TeNT level, and another one has a weak inverse effect. In addition, CodY (control of dciA (decoyinine induced operon) Y) but not Spo0A (sporulation stage 0) or the DNA repair protein Mfd (mutation frequency decline) positively controls TeNT synthesis by interacting with the tent promoter. Moreover, we found that inorganic phosphate and carbonate are among the environmental factors that control TeNT production. Our data show that TeNT synthesis is under the control of a complex network of regulators that are largely distinct from those involved in the control of toxin production in Clostridium botulinum or Clostridium difficile.

Botulinum toxin is a substance produced by Clostridium Botulinum and is responsible for human botulism. This substance is a poison, a neurotoxin, but used in limited quantities it can be a cure for some diseases. It is well connected to a large variety of medical applications. The mechanism of action relies on blocking the acetylcholine at the neuromuscular junction, which blocks the transmission of the nervous impulse with secondary flaccid paralysis. In urology, its role in idiopathic overactive bladder and neurogenic bladder is well known. We performed a thorough review using PubMed and other databases, revising the mechanisms of botulinum toxin action in urologic pathology, treatment procedures and other options. Botulinum toxin is a well-studied substance with a large number of applications in medicine. In urologic pathology, overactive bladder and neurogenic bladder are backed by robust studies that support the therapeutic role of this substance. The toxin has multiple effects, such as inhibition of the nerve growth factor, blocking the bladder sensory afferent pathway and apoptotic effect on the prostate tissue, by inhibiting the substance P, altering the nociceptive pathways. Interstitial cystitis and other rare pathologies show promising results, but further studies are needed. The role of botulinum toxin in benign prostatic hyperplasia is still not elucidated.

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine&ndash, human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD50/100 &micro, g of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 &micro, g) and M4 (1.25 &micro, g), was able to completely neutralize BoNT/B1 (80 i.p. LD50) with a potency greater than 80 i.p. LD50/2.5 &micro, g of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.

Outgrowth and toxinogenesis of Clostridium botulinum Group II (non-proteolytic) type B were studied in cooked ham prepared with different NaNO2 (ranging from 0 to 80 mg/kg) and sodium chloride (NaCl, ranging from 12 to 19 g/kg) incorporation rates. Cured ground pork batters were inoculated with a cocktail of 3 strains of C. botulinum Group II type B at 3.5 log10 CFU/g, portioned and samples of 50 g were vacuum packed then cooked and cooled based on thermal processing employed by the meat processing industry. These cooked ham model samples were stored under reasonably foreseeable conditions of use and storage i.e. for 14 days at 4 °C, followed by a cold chain break for 1 h at 20 °C then up to 33 days at 8 °C. Storage times and temperatures were used to mimic those commonly encountered along the supply chain. Enumeration of C. botulinum and detection of the botulinum neurotoxin type B (BoNT/B) were performed in triplicate at different storage times. Under these experimental conditions, incorporation rates of NaNO2 ≥ 30 mg/kg prevented the outgrowth and toxinogenesis of C. botulinum Group II type B in the cooked ham model, regardless of the NaCl concentrations tested. In contrast, total removal of nitrite allowed outgrowth and toxin production during storage of the processed meat product. Results showed that the maximum ingoing amount of nitrite (i.e. 150 mg/kg) that may be added according to the EU legislation (Regulation (EC) No 1333/2008) can be reduced in cooked ham while still ensuring control of C. botulinum Group II type B. According to the multiple factors that could affect C. botulinum behavior in processing meat products, outgrowth and toxin production of C. botulinum should be evaluated on a case by case basis, depending on the recipe, manufacturing process, food matrix and storage conditions.

Paraquat (PQ) is a widely used herbicide which can cause high mortality to humans. However, relatively few studies focus on metabolic feature of PQ intoxication for investigating the underlying mechanisms. Here we performed non-targeted metabolomics profiling of serum samples from acute and chronic PQ intoxicated mouse models by gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) to identify metabolic feature and characteristic metabolites of acute and chronic PQ intoxication. Results showed that 3-indolepropionic acid (IPA) and pathway of glycine, serine, and threonine metabolism were significantly altered after acute PQ intoxication; 2-hydroxybutyric acid and the ratio of L-serine/glycine were of significance between acute and chronic PQ intoxication. Then targeted metabolomics profiling was conducted by liquid chromatography-mass spectrometry (LC-MS) analysis to confirm the changes of IPA after acute PQ intoxication. Moreover, IPA-producing gut bacteria in feces were quantified by qRT-PCR to explain the varied IPA serum concentration. Clostridium botulinum and Peptostreptococcus anaerobius were significantly suppressed after acute PQ intoxication. The data suggested that PQ caused oxidative damage partially through suppression of anti-oxidative metabolite producing gut bacteria. In conclusion, we identified characteristic metabolites and pathway of acute and chronic PQ intoxication which could be potential biomarkers and therapeutic targets.

A total of 332 staphylococcal strains, mainly isolated from meat, were screened for antibacterial activity. Eighteen strains exhibited antibacterial activity towards species within the same genus. These antibacterial strains were further screened against Clostridium botulinum, to assess their potential as anticlostridial starter cultures for the development of fermented meat products without added nitrate or nitrite. Only Staphylococcus sciuri IMDO-S72 had the ability to inhibit all clostridial strains tested, whilst displaying additional activity against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Apart from their potential as bioprotective cultures, the staphylococcal collection was also screened for biogenic amine production, as these compounds may compromise food quality. To this end, ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was applied. A low incidence of biogenic amine production was found, with tyramine and &beta, phenylethylamine being the most prevalent ones. Concentrations remained relatively low (&lt, 52 mg/L) after a prolonged incubation period, posing no or little threat towards food safety. Taken together, S. sciuri IMDO-S72 could serve as an interesting candidate for the bioprotection of fermented meats as it showed promising antibacterial activity as well as absence of biogenic amine production.

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most potent toxin and produced as a complex with non-toxic components. Food-borne botulism is caused by the ingestion of these BoNT complexes. Hemagglutinin (HA), one of the non-toxic components, is known to have lectin (carbohydrate binding) activity and E-cadherin-binding activity. These activities promote the intestinal absorption of BoNT. To elucidate the mechanism of the onset of food-borne botulism, we focused on the role of HA in the intestinal absorption of BoNT. We describe the functional analysis methods for HA, including the expression of recombinant proteins, binding to glycoproteins and epithelial cells, and localization in mouse intestinal tissue.

Botulinum hemagglutinin (HA) is one of the auxiliary protein components of the botulinum neurotoxin (BoNT) complex, the most lethal toxin known. HA promotes the intestinal absorption of BoNT by at least two mechanisms, resulting in high oral toxicity. One of the mechanisms is the attachment of large progenitor toxin complexes (L-PTCs) to the cell surface of the intestinal epithelium by the carbohydrate-binding activity of HA. The other is epithelial barrier disruption by the E-cadherin-binding activity of HA. The carbohydrate-binding activity of HA also promotes attachment to the basolateral cell surface, which increases the frequency of contact between HA and E-cadherin. Together, the carbohydrate-binding activity of HA is critical for the intestinal absorption of BoNTs. The trimeric triskelion-shaped structure of HA confers the multivalent binding to its ligands and increases the pathogenic biological activities of HA.

Milk inoculated with Clostridium botulinum spores (PA9508B) (107 CFU/mL) was subjected to high-pressure high-temperature treatment (HPHT) (700–900 MPa; 90–110 °C for 0–24 min) using an insulated sample chamber, as well as in a water bath (90–100 °C) (conventional) and surviving spores were enumerated. Destruction kinetics were well described by a first order kinetic model. Conventional heating D values ranged from 156 to 12 min with a z value of 7.8 °C. During HPHT, higher pressures and temperatures resulted in accelerated spore destruction rates. At 900 MPa, the associated D values were 14.5, 1.8 and 0.35 min at 90, 100 and 110 °C, respectively, much lower than the conventional D values. The pressure dependent ZT values were 11.2, 12.3, 12.4 °C at 700, 800, 900 MPa respectively, increasing with pressure. The temperature dependent ZP values were 470, 630, and 800 MPa at 90, 100, and 110 °C, respectively. By comparing ZT with ZP values, it was clear the spores were relatively more sensitive at higher temperatures than at higher pressures in this temperature-pressure range. D value trends beyond 120 °C indicated thermal inactivation was the principal mode of spore destruction and high pressure can protect spores under these conditions.

C.botulinum ATCC 3502 is an obligate rod-shaped spore forming anaerobe causing food poisoning cases worldwide. The increased burden of sporadic hospital outbreaks reflects the pandemicity caused by this pathogen. Several attempts to control the disease surveillance, emergence of antibiotic resistant strains plagued the defense provided by the drugs. Peptidoglycan biosynthesis of C.botulinum ATCC 3502 is targeted to identify potential drug targets due to presence of no functional homolog in host (Homo sapiens). The role of peptidoglycan is to provide strength, fragility and protection to bacterial cells; therefore, it is considered as an attractive target for drug target identification. This study involves systematic exploration of targeted pathway by performing metabolic pathways analysis in two phases (1) mathematical modeling (2) elementary mode analysis (EM). Performing stoichiometric and kinetic modeling discerns the steady state conditions of the system to scrutinize elementary nodes with well-defined objective function. The study identifies Mur ligase enzymes (murA, murB, murC, murD), D-alanine-D-alanine ligase and glutamate racemase potential therapeutic targets for drug discovery. Further, the quantitative analysis characterized their potential in causing the pathogenicity. Physicochemical characterization and subcellular localization analysis assist in understanding biological activity of identified elementary nodes under different environmental conditions.

Background: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. Methods: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. Results: 21 unique BoNT/H LC-HN mAbs were obtained, 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. Conclusions: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis.

We analyzed the volatile compounds produced by serotype D Clostridium botulinum (D-CB16) in trypticase peptone/yeast extract/glucose (TYG) medium using gas chromatography/mass spectrometry (GC/MS). The volatile compounds were captured by solid-phase microextraction and applied to GC/MS for separation and identification of the compounds in TYG medium with or without the cultivation of C. botulinum D-CB16. Thirty-five and 34 volatile compounds were identified in media without and with D-CB16 cultivation, respectively. Of the compounds identified in the medium with the strain, twenty-one were not detected in the original medium, indicating that these were produced by C. botulinum D-CB16. Keywords: Clostridium botulinum, Volatile compounds, Gas chromatography/mass spectrometry, Food poisoning, Botulism

The objective of this study was to investigate the growth kinetics of Clostridium botulinum LNT01, a non-toxigenic mutant of C. botulinum 62A, in cooked ground beef. The spores of C. botulinum LNT01 were inoculated to ground beef and incubated anaerobically under different temperature conditions to observe growth and develop growth curves. A one-step kinetic analysis method was used to analyze the growth curves simultaneously to minimize the global residual error. The data analysis was performed using the USDA IPMP-Global Fit, with the Huang model as the primary model and the cardinal parameters model as the secondary model. The results of data analysis showed that the minimum, optimum, and maximum growth temperatures of this mutant are 11.5, 36.4, and 44.3 °C, and the estimated optimum specific growth rate is 0.633 ln CFU/g per h, or 0.275 log CFU/g per h. The maximum cell density is 7.84 log CFU/g. The models and kinetic parameters were validated using additional isothermal and dynamic growth curves. The resulting residual errors of validation followed a Laplace distribution, with about 60% of the residual errors within ±0.5 log CFU/g of experimental observations, suggesting that the models could predict the growth of C. botulinum LNT01 in ground beef with reasonable accuracy. Comparing with C. perfringens, C. botulinum LNT01 grows at much slower rates and with much longer lag times. Its growth kinetics is also very similar to C. sporogenes in ground beef. The results of computer simulation using kinetic models showed that, while prolific growth of C. perfringens may occur in ground beef during cooling, no growth of C. botulinum LNT01 or C. sporogenes would occur under the same cooling conditions. The models developed in this study may be used for prediction of the growth and risk assessments of proteolytic C. botulinum in cooked meats.