An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production
[['Commonly found in any soil or water environment , the spore forming Gram-positive rod-shaped bacterium Clostridium botulinum and two other clostridia ( C . baratii and C . butyricum ) can , under suitable anaerobic conditions , release botulinum neurotoxins ( BoNTs ) [1 , 2] .', 'BoNTs are highly potent substances with an estimated human lethal dose of ~30-100ng [1 , 2] , and are the most powerful toxins affecting human and animal health .', 'BoNTs cause botulism , a severe neuro-paralytic disease that can lead to death in humans as well as in a range of other mammals and birds [3 , 4] .', 'BoNTs enter into the blood stream in one of three ways: ( 1 ) toxin production by bacteria that have colonized the digestive tract of either children less than 12 months of age ( infant botulism ) or individuals with a suppressed normal intestinal flora ( e . g . , following antibiotic treatment ) including those that have anatomical or functional bowel abnormalities ( Adult intestinal toxemia botulism ) [2 , 5 , 6]; ( 2 ) infection and toxin formation in a wound ( wound botulism ) [5 , 7 , 8]; and ( 3 ) following oral ingestion of pre-formed toxin in contaminated foods ( foodborne botulism ) [2 , 5] .', 'BoNTs target the peripheral motor nerve terminals , blocking neurotransmission by selectively hydrolysing proteins that are involved in the fusion of synaptic vesicles with the presynaptic plasma membrane , thereby preventing acetylcholine release [5 , 6 , 9–11] .', 'Foodborne botulism is a severe and sometimes fatal disease [2] .', 'Although there are fewer cases of foodborne illness caused by C . botulinum than by bacteria of the Salmonella genus , the death rate from botulism is relatively high , 17 . 3 percent , compared with 0 . 5 percent for Salmonella [12] .', 'Thus , the severity of the disease and the widespread presence and persistence of C . botulinum spores make botulism a global health concern and a cause for vigilance [2] .', 'Seven serotypically distinct botulinum neurotoxins ( serotypes A-G ) and more than 40 different subtypes [6 , 9 , 13] are produced by six phylogenetically distinct clostridia ( C . botulinum Groups I-IV and some strains of C . baratii and C . butyricum ) .', 'Considering the highly potent nature of the neurotoxin , methods that limit the proliferation of C . botulinum and the associated neurotoxin production in food are a major priority for the food-processing industry; these processes are complicated by the physiological differences among clostridia .', 'The structures and the mechanisms of action of BoNTs are reasonably well established [11 , 14–20] , however , regulation of botulinum neurotoxin gene ( bont ) expression and BoNT production are not fully understood .', 'Likewise , the environmental signals which trigger the synthesis of the BoNTs and the regulatory network and actors which control the production of the toxin ( and many subsequently regulated genes ) remain to be elucidated .', 'What is known , firstly , is that in vitro experimental methods developed and applied to monitor bont gene expression in C . botulinum show a peak in neurotoxin gene expression during late exponential and early stationary phase of population growth; expression decreases drastically during stationary phase for C . botulinum Group I type A [21–26] and for C . botulinum Group II type E [22 , 27] .', 'However some of these studies examined a relatively small number of time points during population growth so that the full bont gene expression profile is not always observed .', 'Moreover , these studies show that the quantity of BoNT produced can be influenced by the strain , by culture conditions and by the nutritional status of the medium—although the precise mechanisms are unknown .', 'Secondly , bont gene expression is reported to be tightly regulated through positive and negative regulatory systems .', 'Positively , through the participation of BotR [17] , Agr quorum sensing system [28] , CodY [29] and CLC_1094/CLC_1093 ( equivalent to CBO_1042/CBO_1041 ) , CLC_1913/ CLC_1914 ( equivalent to CBO_1967/ CBO_1968 ) and CLC_0663/CLC_0661 ( equivalent to CBO_0608/CBO_0607 ) two component signal transduction systems [30] .', 'Negatively , through the participation of CBO0787/CBO0786 ( equivalent to CLC_0843/CLC_0842 ) [31] which is also a two component signal transduction system [32] .', 'Thirdly , in most C . botulinum Group I type A1 strains , the genes encoding the neurotoxin ( bont ) and its associated non-toxic neurotoxin proteins ( ANTPs ) ( ntnh , has ) are located in a gene cluster and are organized in two transcriptional units ( or operons ) , namely , the ntnh-bont and ha operons [9 , 33] .', 'The first operon ( ntnh-bont ) , which is located at the 3′ end of the botulinum locus , encompasses the bont gene immediately preceded by the ntnh gene .', 'Both genes are co-transcribed in the same orientation , and the organization of this operon is highly conserved in all botulinum toxin forming clostridia .', 'The second operon contains the ha genes and differs slightly between the various subtypes ( BoNT/A1 , A5 , B , C , D and G ) .', 'The ha operon contains successive genes for the 33 kDa ( ha33 ) , 17 kDa ( ha17 ) , and 70 kDa ( ha70 ) hemagglutinins [30 , 34] .', 'These hemagglutinin genes are localised upstream of the ntnh-bont genes and are transcribed in the opposite orientation [5 , 35 , 36] .', 'Thus , the nontoxic proteins for subtype A1 include NTNHA ( which together with BoNT forms the minimally functional progenitor toxin complex ( M-PTC ) ) and three hemagglutinin ( HA ) proteins ( HA70 , 17 and 33 ) , which assemble ( with the M-PTC ) to form the large size toxin complex ( L-PTC ) [10 , 37 , 38] .', 'Lastly , BoNT is released from the bacterium and exists in nature in the form of a complex [36 , 39 , 40] , i . e . not as a pure toxin [41] .', 'The distinct neurotoxins form complexes of different sizes ( from 288 to 900 kDa ) by association with ANTPs , i . e . hemagglutinins ( HAs ) and nontoxic non-hemagglutinins ( NTNHs ) .', 'These ANTPs spontaneously associate with BoNTs at low pH and dissociate at pH 7 . 5 and above .', 'The associated proteins protect the neurotoxin and facilitate its absorption into the body [37 , 42] .', 'C . botulinum Group I type A1 ( BoNT/A1 ) neurotoxins are so far the best characterized neurotoxins , a consequence of both their frequent involvement in human botulism worldwide as well as their greater potency and , therefore , suitability for therapeutics [1] .', 'With all the aforementioned findings , it is reasonable to conclude that bont gene transcription and neurotoxin production may be influenced by the bacterial strain .', 'In particular gene transcription may be influenced by the availability of particular nutrients ( although the precise mechanisms are unknown ) that are present during the transition from late-exponential to early-stationary phase cultures ( i . e . , growth phase dependent ) .', 'In turn this transcription is dependent on both positive and negative regulatory elements .', 'This evidence supports the construction of a signal transduction and sensory transcription regulatory network to describe the kinetics of neurotoxin production [32] .', 'Current mathematical models of C . botulinum are based on statistical data aggregation and describe beliefs concerning the unknown concentrations of C . botulinum spores in the environment , the uncertain inactivation kinetics for populations of spores at high temperatures and the germination and growth of C . botulinum populations for a variety of physico-chemical conditions [26 , 43–51] .', 'These models do not attempt to identify elements of regulatory control which are the key to transferability and to an appreciation of cell to cell variations ( in many situations foodborne botulism may be driven by very few cells so that cell variability is a crucial unknown ) .', 'Furthermore current models sum-up many component processes , such as signalling , permeability and enzymatic activity , obscuring opportunities for improved understanding .', 'The use of computational models amenable to simulation and to the analysis of what-if type scenarios would permit further formulation of hypotheses concerning the gene expression profiles and interactions; additionally a process of iterative computer simulation would guide future experimentation .', 'In this report we tackle the challenge of integrating the various sources of multi-scale biological evidence into a mechanistic model using ordinary differential equations .', 'In turn this model is used to explain the regulated toxigenesis process of C . botulinum Group I type A1 .', 'Strains of C . botulinum Group I type A1 fall into three classes ,', '( i ) those that carry the neurotoxin gene in an ha cluster ,', '( ii ) those that carry the neurotoxin gene in an orfx cluster , and', '( iii ) those that also carry a type B neurotoxin gene and form a small amount or no type B neurotoxin .', 'We focus on the first of these , strains that carry the neurotoxin gene in an ha cluster [9 , 52] .', 'We use biological data from the literature that relates to five strains of C . botulinum Group I type A1 ( ATCC19397 , 62A , Hall A , Hall A-hyper and ATCC3502 ) ; the close relationship between these strains having been established by whole genome sequencing , microarray analysis and MLST [53–56] .', 'Additionally , these five strains all possess identical or very similar bont and botR genes [52 , 57 , 58] .', 'This modelling task , to the best of our knowledge , has not been approached so far .', 'We first review the experimental knowledge that has been published in the literature on the patterns of toxin production and toxin gene expression and then identify the main aspects of the regulation , highlighting the key molecular players .', 'The main contribution of this report is shown in the results section where the diverse available information used in constructing the mathematical model of toxin formation by C . botulinum Group I type A1 is integrated into a complete model in an incremental way .', 'The proposed model is implemented and simulated , to confirm its ability to reproduce the observed patterns of behaviour , in various wild type [WT] strains and in various mutant strains that have previously been experimentally characterized .', 'We further discuss the results and focus on a review of the hypotheses that were made throughout the modelling process , identifying the opportunities they offer for the definition of specific experimental settings that would help in shedding light on several of the poorly understood steps in the process of toxin formation by C . botulinum Group I type A1 .'], ['The proposed mathematical model is based on a continuous-deterministic approach , where the components of the model , such as concentrations , number of bacterial cells etc . , are represented as continuous variables and their variation over time is expressed through their first derivative .', 'The dynamics of the multi-component system corresponds with a set of coupled ordinary differential equations for which numerical solutions are obtained by computer simulation .', 'To avoid dealing with the mathematical details of differential equations , we adopted a reaction-based specification language to describe the interactions among the model variables .', 'The whole modelling process is supported by the COPASI modelling and simulation software package [62] , which takes as its input the reaction-based specification of the model , and provides the simulated time courses of the variable dynamics .', 'To simplify the process of model definition , we used an incremental procedure which allowed us to build increasingly complex versions of the model , each one incorporating additional pieces of biological evidence and some additional modelling assumptions .', 'The modelling approach considers two separate levels of representation: ( 1 ) at the cell level , the dynamics of the population in a culture and ( 2 ) at a sub-cellular level , the network that regulates toxigenesis and gene expression .', 'At a cell level the model describes the dynamics of the consumption of nutrients and of quorum-sensing signals in the culture .', 'This is then coupled with the dynamics of the regulation and gene-expression which is described by the sub-cellular level .'], ['Details of a computational model for the growth of a population of C . botulinum Group I type A1 cells in a culture have been reported by [44] .', 'As thoroughly explained in [44] , the rationale underlying the need of modelling population dynamics is rooted in the experimentally observed correlation between the bacterial growth phase and the toxin production process .', 'Further evidence supporting this correlation at genetic regulation level is described in the section on the molecular model of BoNT synthesis regulation .', 'The mathematical modelling of C . botulinum cultures is based on a compartmentalization of the growing population of cells into three distinct groups: The initial population of C . botulinum cells is fully composed of AC cells , which later evolve to RCs and may commit to sporulation and become SCs .', 'These processes are influenced by some biochemical species generically termed “Signal” , as shown in Fig 1 .', 'A future development , not currently included , is to extend the present analysis to start with bacterial spores and to therefore incorporate steps for spore germination and outgrowth [64] .', 'Different hypotheses relating to the nature of the “signal ( s ) ” ( previously described in [44] ) led to the discrimination of plausible modelling scenarios and were used to generate corresponding models that were then evaluated on their ability to reproduce the observed pattern of growth observed for C . botulinum type A1 strain ATCC 19397 .', 'We found that a model where two distinct signal sources were considered—the first one determined by the abundance of nutrients essential to C . botulinum cell growth , which we denoted by the abstract species N , and a second one endogenously produced by the bacterial cells and used as a quorum-sensing signal , denoted by S–was most successful at explaining the pattern of growth observed for C . botulinum type A1 strain ATCC 19397 .', 'A diagrammatic representation of this modelling option is included in Fig 1 .', 'In this model the rate of cell reproduction increases with the nutrient concentration , N , whilst the rate of sporulation increases with the concentration of the chemical signal S . The model proposed in [44] was encoded using eight reactions .', 'We consider here an updated version , still based on the same rationale , which is encoded by the six reactions listed in Table A of Supporting Information File 1 ( Table A in S1 Text ) .', 'As previously reported in Figure 6 of [44] , this model produces a good fit for the experimental growth data generated for strain ATCC 19397 .', 'Several environmental stimuli have been identified with positive and negative regulation of toxin production in C . botulinum Group I type A1 .', 'Neurotoxin production has been reported to be associated with the transition from late-exponential to early-stationary phase cultures .', 'This is indicated by a peak in the level of neurotoxin gene cluster expression that is clearly observable in the late-exponential to early-stationary phase of cultures and which drastically decreases during the later stationary phase ( as shown in Fig 2 ) .', 'Moreover , the expression patterns for all the genes , in both the ntnh/bont and the ha operon , show an equivalent correlation with population dynamics ( data available in [26] , [21] , [22] and [23] ) .', 'This points to regulatory elements that link population growth to toxigenesis in C . botulinum type A1 .', 'In this section we expound the procedure used for calibrating model parameters , and then proceed to validate the model by checking against characteristics of toxigenesis which have been reported previously in sections on nutrient and quorum-sensing regulated population growth model and the molecular model of BoNT synthesis regulation .', "To find suitable values for model parameters , we used the experimental data from [59] for type A1 strain ATCC 19397 , which we considered as the 'wild type' organism for the purpose of our modelling ( WT , hereafter ) .", 'The experimental time course for the population size ( measured in CFU/ml over time in [59] ) provides the parameters of the population sub-model , i . e . the kinetic parameters of reactions ( 1 ) to ( 6 ) provided in Table A of Supporting Information File 1 ( Table A in S1 Text ) .', 'The amount of toxin in the supernatant measured in the same experiment in [59] ( measured in MLD50/ml over time ) provides the data for fitting the gene expression sub-model , i . e . the kinetics of reactions ( 1 ) to ( 49 ) listed in Table B of Supporting Information File 1 ( Table B in S1 Text ) .', 'The model parameters are reported in Supporting Information File 2 ( S2 Text ) .', 'The fitted model is compared with the WT experimental data in Fig 5A and 5B .', 'The experimental data points are shown as empty circles , whereas the computational model is reported as continuous lines .', "There is an inevitable match between model outcomes and the experimental 'model training' data , which is confirmed by analysis of correlation .", 'For the population an R2 measure is 0 . 975 while for the toxin production it is 0 . 95 .', 'After tuning model parameters to fit WT observed behaviour , we proceeded to validate the model , by assessing its ability to reproduce the behaviours experimentally observed in the different C . botulinum mutant strains we had considered in the study .', 'We examined four different mutations , which are implemented in the WT model exclusively by changing the initial state of the model , i . e . without any change to the kinetics of the reactions .', 'The mutants we considered for the purposes of our validation are as follows: For each mutant , we obtain and report the toxigenesis predictions ( pattern and amount of BoNT ) from both the WT model and the mutant model .', 'Then we examine the relationship between model predictions and the experimental results to determine the ability of the models to reproduce wet-lab evidence .'], ['For the first time we have defined and implemented a computational model , at the molecular level , for the highly regulated process of BoNT production in C . botulinum Group 1 type A1 .', 'In contrast to existing modelling approaches , largely aimed at risk assessment for C . botulinum , this development does not integrate out component processes such as signalling , membrane permeability and metabolic activity , and it does include elements of genetic in-formation .', 'The model captures causal relations among the known regulators of toxigenesis , at the molecular level .', 'This leads to a computational model which is able to embrace both the population dynamics of the cells ( so that we were able to include growth phase-dependent patterns of bacterial behaviour ) as well as behaviour of the genetic regulatory network and the molecular interactions that link toxin expression with the environmental and population generated signals .', 'The model construction has integrated the available experimental knowledge on the factors that , at a molecular level , regulate toxigenesis in C . botulinum Group 1 type A1; previously reviewed in [32] .', 'In addition it portrays the effects of nutrient availability and quorum-sensing molecules and their coupling with distinct sensing and response TCSs that are regulators that mediate the activation of the toxin coding genes .', 'The model satisfies a validation based on its ability to predict the effects of various mutations that have been experimentally studied in vitro .', 'The validation results suggest that the model is able to provide a plausible explanation for the interplay of the multiple regulation mechanisms that impact toxin production in C . botulinum Group 1 type A1 .', 'Models that encode causality have significant advantages over purely statistical descriptions , because they lend themselves to exploration of what-if-scenarios and generating test-able hypotheses .', 'For instance , the model proposed here can be used to predict the pheno-types of mutants that have not yet been studied in vitro .', 'As an example , we can explore the predicted toxin production of a mutant cell where the positive regulator CodY is removed and also the negative regulator TCS CBO0787/CBO0786 is silenced ( the CO-DY_M+C786_M double mutant , based on the abbreviation used in the section on the computational model ability to reproduce additional experimental results .', 'We can then compare the predicted concentration of toxin in the supernatant for this double mutant with that obtained by the C . botulinum WT model ( Fig 9 ) .', 'Our model predicts that silencing the CBO0787/CBO0786 TCS rescues the CODY_M mutant ability to produce toxin , to levels similar to those of the wild type .', 'This is a prediction that can be tested in vitro , to provide either further support for the model structure , or to generate new evidence that can be integrated into parameterization and hence improve the predictive capability .', 'Furthermore , models can be tested for conditions not yet considered in the laboratory set-ting; thus obtaining additional predictions that could be conductive to the definition of experimental settings .', 'This novel model is an initial attempt to elucidate toxigenesis in C . botulinum Group 1 type A1 .', 'We expect it will require further tuning , improvements and changes .', 'We made a substantial number of assumptions about the dynamics of the activation of promoters , which require experimental confirmation .', 'The process of toxigenesis has been simplified not to include too many unknown details of the hemagglutinins and NTNH synthesis , together with the complexation process that generates the functional forms of the toxin .', 'Clearly it is essential that the amount and quality of experimental results is increased .', 'In the absence of large datasets on a specific genotype , we had to construct the model from experimental data obtained from varying , though closely related , strains .', 'Each study used a different granularity for data collection and a distinct measurement technique , which gave us the challenging task of validating a quantitative model with qualitative data .', 'Continuing with improving the reliability of model predictions and refining the model with the inclusion of additional experimental evidence is the subject of our on-going research work .']]
['Introduction', 'Materials and Methods', 'Results', 'Discussion and Conclusions']
['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 .']
['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 .', 'In this paper we present modelling based on the integration of diverse information 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 .', 'Here , we integrate these regulatory elements into a combined model of population dynamics and gene regulation to build the 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 that could be used to prevent botulism , and potentially contribute to improved methods for the production of toxin used for therapeutics .']
['clostridium botulinum', 'medicine and health sciences', 'toxins', 'gut bacteria', 'pathology and laboratory medicine', 'gene regulation', 'operons', 'neurotoxins', 'toxicology', 'toxic agents', 'neurotoxicology', 'dna', 'bacteria', 'bacterial toxins', 'gene expression', 'biochemistry', 'predictive toxicology', 'nucleic acids', 'genetics', 'biology and life sciences', 'botulinum toxin', 'organisms']