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.

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.

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.

The influence of glyphosate on the microbiota and production of botulinum neurotoxin during ruminal fermentation.

The aim of the present study is to investigate the impact of glyphosate on the microbiota and on the botulinum neurotoxin (BoNT) expression during in vitro ruminal fermentation. This study was conducted using two DAISY(II)-incubators with four ventilated incubation vessels filled with rumen fluid of a 4-year-old non-lactating Holstein-Friesian cow. Two hundred milliliter rumen fluid and 800 ml buffer solution were used with six filter bags containing 500 mg concentrated feed or crude fiber-enriched diet. Final concentrations of 0, 1, 10, and 100 µg/ml of glyphosate in the diluted rumen fluids were added and incubated under CO2-aerated conditions for 48 h. The protozoal population was analyzed microscopically and the ruminal flora was characterized using the fluorescence in situ hybridization technique. Clostridium botulinum and BoNT were quantified using most probable number and ELISA, respectively. Results showed that glyphosate had an inhibitory effect on select groups of the ruminal microbiota, but increased the population of pathogenic species. The BoNT was produced during incubation when inoculum was treated with high doses of glyphosate. In conclusion, glyphosate causes dysbiosis which favors the production of BoNT in the rumen. The global regulations restrictions for the use of glyphosate should be re-evaluated.

A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins.

BACKGROUND: Clostridium botulinum strain IBCA10-7060, isolated from a patient with infant botulism, produced botulinum neurotoxin type B (BoNT/B) and another BoNT that, by use of the standard mouse bioassay, could not be neutralized by any of the Centers for Disease Control and Prevention-provided monovalent polyclonal botulinum antitoxins raised against BoNT types A-G. METHODS AND RESULTS: The combining of antitoxins to neutralize the toxicity of known bivalent C. botulinum strains Ab, Ba, Af, and Bf also failed to neutralize the second BoNT. Analysis of culture filtrate by double immunodiffusion yielded a single line of immunoprecipitate with anti-A, anti-B, and anti-F botulinum antitoxins but not with anti-E antitoxin. A heptavalent F(ab')2 botulinum antitoxin A-G obtained from the US Army also did not neutralize the second BoNT. An antitoxin raised against IBCA10-7060 toxoid protected mice against BoNT/B (Okra) and against the second BoNT but did not protect mice against BoNT/A (Hall) or BoNT/F (Langeland). CONCLUSION: The second BoNT thus fulfilled classic criteria for being designated BoNT/H. IBCA10-7060 is the first C. botulinum type Bh strain to be identified. BoNT/H is the first new botulinum toxin type to be recognized in >40 years, and its recognition could not have been accomplished without the availability of the mouse bioassay.

Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

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.

Clostridiolysin S, a post-translationally modified biotoxin from Clostridium botulinum.

Through elaboration of its botulinum toxins, Clostridium botulinum produces clinical syndromes of infant botulism, wound botulism, and other invasive infections. Using comparative genomic analysis, an orphan nine-gene cluster was identified in C. botulinum and the related foodborne pathogen Clostridium sporogenes that resembled the biosynthetic machinery for streptolysin S, a key virulence factor from group A Streptococcus responsible for its hallmark beta-hemolytic phenotype. Genetic complementation, in vitro reconstitution, mass spectral analysis, and plasmid intergrational mutagenesis demonstrate that the streptolysin S-like gene cluster from Clostridium sp. is responsible for the biogenesis of a novel post-translationally modified hemolytic toxin, clostridiolysin S.

Clostridium botulinum in the post-genomic era.

Foodborne botulism is a severe neuroparalytic disease caused by consumption of botulinum neurotoxin formed by strains of proteolytic Clostridium botulinum and non-proteolytic C. botulinum during their growth in food. The botulinum neurotoxin is the most potent substance known, with as little as 30-100 ng potentially fatal, and consumption of just a few milligrams of neurotoxin-containing food is likely to be sufficient to cause illness and potentially death. In order to minimise the foodborne botulism hazard, it is necessary to extend understanding of the biology of these bacteria. This process has been recently advanced by genome sequencing and subsequent analysis. In addition to neurotoxin formation, endospore formation is also critical to the success of proteolytic C. botulinum and non-proteolytic C. botulinum as foodborne pathogens. The endospores are highly resistant, and enable survival of adverse treatments such as heating. To better control the botulinum neurotoxin-forming clostridia, it is important to understand spore resistance mechanisms, and the physiological processes involved in germination and lag phase during recovery from this dormant state.

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.

Detection and confirmation of Clostridium botulinum in water used for cooling at a plant producing low-acid canned foods.

Our laboratory tested water samples used for cooling low-acid canned foods at a canning facility under investigation by the U.S. Food and Drug Administration. We used an enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies (DIG-ELISA) and real-time PCR as screening methods and confirmed the presence of neurotoxin-producing Clostridium botulinum in the samples by mouse bioassay.

Effects of carbon dioxide on growth of proteolytic Clostridium botulinum, its ability to produce neurotoxin, and its transcriptome.

The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO2 had little effect on any of these parameters. At all CO2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.

Clostridium botulinum in cattle and dairy products.

The use of plastic-wrapped and nonacidified silage as cattle feed has led to an increasing number of botulism outbreaks due to Clostridium botulinum Groups I-III in dairy cattle. The involvement of Groups I and II organisms in cattle botulism has raised concern of human botulism risk associated with the consumption of dairy products. Multiplication of C. botulinum in silage and in the gastrointestinal tract of cattle with botulism has been reported, thus contamination of the farm environment and raw milk, and further transmission through the dairy chain, are possible. The standard milk pasteurization treatment does not eliminate spores, and the intrinsic factors of many dairy products allow botulinal growth and toxin production. Although rare, several large botulism outbreaks due to both commercial and home-prepared dairy products have been reported. Factors explaining these outbreaks include most importantly temperature abuse, but also unsafe formulation, inadequate fermentation, insufficient thermal processing, post-process contamination, and lack of adequate quality control for adjunct ingredients were involved. The small number of outbreaks is probably explained by a low incidence of spores in milk, the presence of competitive bacteria in pasteurized milk and other dairy products, and growth-inhibitory combinations of intrinsic and extrinsic factors in cultured and processed dairy products.

Science, practice, and human errors in controlling Clostridium botulinum in heat-preserved food in hermetic containers.

The incidence of botulism in canned food in the last century is reviewed along with the background science; a few conclusions are reached based on analysis of published data. There are two primary aspects to botulism control: the design of an adequate process and the delivery of the adequate process to containers of food. The probability that the designed process will not be adequate to control Clostridium botulinum is very small, probably less than 1.0 x 10(-6), based on containers of food, whereas the failure of the operator of the processing equipment to deliver the specified process to containers of food may be of the order of 1 in 40, to 1 in 100, based on processing units (retort loads). In the commercial food canning industry, failure to deliver the process will probably be of the order of 1.0 x 10(-4) to 1.0 x 10(-6) when U.S. Food and Drug Administration (FDA) regulations are followed. Botulism incidents have occurred in food canning plants that have not followed the FDA regulations. It is possible but very rare to have botulism result from postprocessing contamination. It may thus be concluded that botulism incidents in canned food are primarily the result of human failure in the delivery of the designed or specified process to containers of food that, in turn, result in the survival, outgrowth, and toxin production of C. botulinum spores. Therefore, efforts in C. botulinum control should be concentrated on reducing human errors in the delivery of the specified process to containers of food.