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

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

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

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

Effect of Fill Temperature on Clostridium botulinum Type A Toxin Activity during the Hot Filling of Juice Bottles.

The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum (BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinum type A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinum type A acid mud toxin (∼10(6) mouse lethal dose [MLD50]/ml) was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles (12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min) by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle's bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 10(6) MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 10(6) MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 10(5) MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 10(6) MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 10(4) MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

An electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of Clostridium botulinum outbreak strains associated with type A botulism.

BACKGROUND: Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products. RESULTS: The limit of detection of this ECL assay is 40 pg mL⁻¹ for BoNT/A complex, 10 pg mL⁻¹ for SEB and 40000 CFU mL⁻¹ for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. CONCLUSION: The results of the study indicate that this ECL assay is very sensitive, rapid (<6 h) and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays.

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

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

The fur transcription regulator and fur-regulated genes in Clostridium botulinum A ATCC 3502.

Clostridium botulinum is a spore-forming bacterium that can produce a very powerful neurotoxin that causes botulism. In this study, we have investigated the Fur transcription regulators in Clostridium botulinum and Fur-regulated genes in Clostridium botulinum A ATCC 3502. We found that gene loss may be the main cause leading to the different numbers of Fur transcription regulators in different Clostridium botulinum strains. Meanwhile, 46 operons were found to be regulated by the Fur transcription regulator in Clostridium botulinum A ATCC 3502, involved in several functional classifications, including iron acquisition, iron utilization, iron transport, and transcription regulator. Under an iron-restricted medium, we experimentally found that a Fur transcription regulator (CBO1372) and two operons (DedA, CBO2610-CBO2614 and ABC transporter, CBO0845-CBO0847) are shown to be differentially expressed in Clostridium botulinum A ATCC 3502. This study has provided-us novel insights into the diversity of Fur transcription regulators in different Clostridium botulinum strains and diversity of Fur-targeted genes, as well as a better understanding of the dynamic changes in iron restriction occurring in response to this stress.

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

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

Stability of the Clostridium botulinum type A neurotoxin complex: an empirical phase diagram based approach.

Clostridium botulinum type A neurotoxin (BoNT/A complex) is of great interest to the pharmaceutical industry. The drug itself is a natural complex of the toxin and a number of associated proteins. Surprisingly, relatively little is known about the exact structure and stability of the 900 kDa BoNT/A complex and its component proteins with the exception of the 150 kDa neurotoxin. In this study we describe the relative stability of the BoNT/A complex, the neurotoxin, and its associated proteins over a wide range of temperature and pH employing circular dichroism, intrinsic and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and static light scattering. The data suggest a strong stabilizing effect of the associated proteins on the neurotoxin component. This data is compiled into empirical phase diagrams which permit the simultaneous visualization of multiple data sets over a wide range of conditions.

Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR.

Production of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the botR/A, bont/A and antp genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of Clostridium botulinum A. The botR/A expression level was about 100-fold less than those of the bont/A and antp genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the botR/A, bont/A and antp genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 degrees C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 degrees C. High temperature did inhibit transcription, since expression levels of the botR/A, bont/A and antp genes were similar in cultures at 37 and 44 degrees C. However, incubation at 44 degrees C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In C. botulinum E, which contains no gene related to botR, the bont/E and p47 genes were also expressed during the transition phase, and no protease activation at 44 degrees C was evident.

Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains.

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.