- Research article
- Open Access
Epigenetic acquisition of inducibility of type III cytotoxicity in P. aeruginosa
© Filopon et al; licensee BioMed Central Ltd. 2006
- Received: 25 November 2005
- Accepted: 30 May 2006
- Published: 30 May 2006
Pseudomonas aeruginosa, an opportunistic pathogen, is often encountered in chronic lung diseases such as cystic fibrosis or chronic obstructive pneumonia, as well as acute settings like mechanical ventilation acquired pneumonia or neutropenic patients. It is a major cause of mortality and morbidity in these diseases. In lungs, P. aeruginosa settles in a biofilm mode of growth with the secretion of exopolysaccharides in which it is encapsulated, enhancing its antibiotic resistance and contributing to the respiratory deficiency of patients. However, bacteria must first multiply to a high density and display a cytotoxic phenotype to avoid the host's defences. A virulence determinant implicated in this step of infection is the type III secretion system (TTSS), allowing toxin injection directly into host cells. At the beginning of the infection, most strains isolated from patients' lungs possess an inducible TTSS allowing toxins injection or secretion upon in vivo or in vitro activation signals. As the infection persists most of the bacteria permanently loose this capacity, although no mutations have been evidenced. We name "non inducible" this phenotype. As suggested by the presence of a positive feedback circuit in the regulatory network controlling TTSS expression, it may be due to an epigenetic switch allowing heritable phenotypic modifications without genotype's mutations.
Using the generalised logical method, we designed a minimal model of the TTSS regulatory network that could support the epigenetic hypothesis, and studied its dynamics which helped to define a discriminating experimental scenario sufficient to validate the epigenetic hypothesis. A mathematical framework based on formal methods from computer science allowed a rigorous validation and certification of parameters of this model leading to epigenetic behaviour. Then, we demonstrated that a non inducible strain of P. aeruginosa can stably acquire the capacity to be induced by calcium depletion for the TTSS after a short pulse of a regulatory protein. Finally, the increased cytotoxicity of a strain after this epigenetic switch was demonstrated in vivo in an acute pulmonary infection model.
These results may offer new perspectives for therapeutic strategies to prevent lethal infections by P. aeruginosa by reverting the epigenetic inducibility of type III cytotoxicity.
- Positive Feedback Circuit
- Computation Tree Logic
- Formal Sentence
- Strain PAO1
- Secretory Phenotype
Epigenetic phenotypic modifications arise and can be transmitted from a cell to its progeny in the absence of any genetic modifications. As a consequence, several phenotypes may arise from the same genome in the same conditions, which is equivalent to the existence of multiple steady states according to physicist's terminology. Such a phenomenon has been under investigations for example in the lactose metabolic network of Escherichia coli since 1957 [10–12]. Since all the genes necessary for cytotoxic secretion are present and can be activated in non inducible P. aeruginosa strains, this is consistent with the hypothesis that the transition from inducibility to non inducibility of type III cytotoxicity is an epigenetic switch. Thomas conjectured that a positive feedback circuit (comprising interacting elements, each of which exerts, directly or indirectly, a positive action on itself) in a non linear dynamical system, such as a regulatory network controlling a biological process, is a necessary condition (although not sufficient) for the existence of multiple steady states[13, 14]. This hypothesis has now been formally demonstrated[15, 16]. Thus, the presence of the positive feedback circuit in the TTSS regulatory network where ExsA positively regulates it's own synthesis, could lead to a bistable behaviour for the gene exsA (either on or off) corresponding to an epigenetic switch of the TTSS (inducible or not). However, at least two intertwined feedback circuits control the production of ExsA: a positive feedback circuit at the transcriptional level (ExsA is required for its own synthesis) and a negative feedback circuit (ExsA is required for the synthesis of its inhibitor) (Figure 1B). The existence of the positive circuit is a necessary, not sufficient, condition for multistationarity and more particularly epigenetic bistable switches. Consequently, one has to check if the system actually displays multistationarity. Molecular techniques can not by themselves prove or refute this hypothesis. Indeed, the absence of a mutation can be very difficult to demonstrate, since all possible genes in which a mutation could induce such a phenotypic change may not be known. Furthermore, the presence of a mutation in a putatively involved gene in inducible strains may not mean that it is the initial cause of the phenotypic change: an epigenetic switch could be the first event, followed by a mutation that may further stabilize it. Mathematical analysis of the network's dynamic is therefore necessary to determine if the epigenetic hypothesis is coherent: this means if a reasonable model, which exhibits two stable solutions, can be drawn. As the parameters of the system are not known, we used generalized logics, a method derived from a Boolean approach[18, 19].
The actual regulatory network to analyse is the one in figure 1A. However, proteins ExsC and ExsE are modulators of protein ExsD activity, and can be omitted from the model (Figure 1B) as far as the aim of this model is not to describe the whole regulatory network, but only to address the question of the possibility of an epigenetic switch between inducibility and non inducibility of the TTSS. Figure 1C shows the graph that models the minimal regulatory network: vertices represent the biological entities (x = ExsA, and y = ExsD) and lines their interactions ("-" for negative action and "+" for positive action). The output (z) is the production of the secretory devices and toxins secretion under induction by calcium depletion, i.e. inducibility. This graph is similar to the one worked out for the regulation of mucoidy in the same bacteria. This model, a formal computer science approach, using model checking Computation Tree Logic (CTL) and a dedicated framework SMBioNet, allowed us to fully establish the consistency of the epigenetic hypothesis for the acquisition of a type III cytotoxicity and to propose experimental evidences that are sufficient to prove it. We thus could design in vivo and in vitro experiments to test it.
Generalised logical analysis
Another aspect of the generalised logical method is that it allows analysis of the dynamic properties of a network in terms of the "functionality" of its constitutive feedback circuits. The effect of a circuit does not only depend on the mere existence of the relevant interactions, but also on their relative strengths, which are noted as discrete parameters (K). For a given regulatory network, the number of dynamics depends on the number of thresholds and parameters. In the case of the two graphs depicted in figure 2, there are 324 different combinations of parameters for each graph. Each set of parameters defines a specific temporal behaviour. Different set of values of the parameters can lead or not to epigenetic behaviour.
The use of a formal computer science method allows to select parameter set that lead to a model exhibiting the hypothesised behaviour
Finding suitable valuations for parameters constitutes a major issue for the modelling. We runned the whole corpus of formal methods from computer science to include the dynamical knowledge or hypotheses on the system. Our method starts by expressing the biological hypothesis (i.e. the epigenetic behaviour) formally, as formal sentences which can be manipulated automatically by computer. Here we applied a widely used temporal logic called Computation Tree Logic, because time plays a central role in behavioural properties. Moreover, formal sentences in CTL can be automatically checked against the 648 models by using a Model Checking algorithm. Technically, behaviour is represented by a transition system (state graph), which can be automatically computed from the regulatory network and the values of parameters. Then, it becomes possible to extract (via the 'brut force technique') the sets of parameters, if any, which lead to a behaviour that satisfies the CTL sentences. Model Checking thereby classifies the set of possible dynamic behaviours into two groups: those which satisfy the property and those which do not. This methodology is instrumented by the software SMBioNet  which, for the given regulatory network, automatically returns all sets of parameters which make the hypothesis coherent with the model.
Design of a discriminating experimental scenario
In addition to proving the consistency of the epigenetic hypothesis, formal methods can establish the discriminating power of the experimental scenario. The goal is to prove both formal sentences in vivo. The two previous formulae reflect the existence of two steady states. As indicated above, the first formula states that the basal level of x is a steady state and is consequently satisfied in vivo which is true for a non inducible strain. Thus, only the second formula remains to be experimentally validated. The second formal sentence ((x = 2) = > Fs (z = 1)) is always true when x ≠ 2 because, according to the truth table, the implication (false = > anything) is always true. Consequently it is unnecessary to conduct an experiment where x ≠ 2. Therefore any experiment must start by pushing the concentration of x to 2 which means to pulse x to saturation with an external signal. Moreover Fs(z = 1) signifies "wait a lapse of time to allow the system to settle down and check if the bacterium has acquired a cytotoxic phenotype". If the bacterium has not changed its phenotype, then the experiment a priori fails. Consequently the second part of the experimental scenario is rigorously sufficient to check the second formal sentence. The length of this "lapse of time" must be determined experimentally; here it stands for "as many generations later as possible". If the bacterium has acquired an inducible phenotype, that lasts at least several generations after the external signal has been removed, then epigenesis is proven.
This formal approach was useful to suggest in vitro and in vivo experiments to test our hypothesis. It showed that the two stable attractors differ only by the amount of ExsA protein, and that, if the hypothesis is correct, no differences in the concentration of the inhibitor ExsD can lead to a change in phenotype (Figure 3B). Thus, if a transient increase in the amount of ExsA protein can shift the system from a non cytotoxic to a stable cytotoxic state, the existence of the epigenetic switch would be demonstrated.
An ExsA pulse allows to recover a stable type III secretory phenotype
Experiments suggested by the formal method require pulsing ExsA by application of an external stimulus and observing the change in phenotype (if any) and its stability when the stimulus is removed. In order to pulse ExsA, we added an additional exsA gene, under the control of an inducible promoter, to a non inducible P. aeruginosa strain PAO1. Thus, we constructed plasmid pexsA ind. In this construction, transcription of exsA is under the control of the ptac promoter repressed by the LacI protein produced in large amounts because of the presence of the lacI q gene. Under inducing conditions (presence of isopropyl-beta-D-thiogalactopyranoside, IPTG), transcriptional repression by LacI is inhibited, permitting over-expression of the exsA gene from the ptac promoter. This induction is immediately released when the medium is depleted of the inducer. The most straightforward test to determine the resulting phenotype is the electrophoretic detection of the type III toxins secreted in vitro after bacteria have been submitted to calcium depletion.
PAO1 (pexsA ind) acquires an in vivo type III cytotoxicity after a transient increase of ExsA
Despite a functional wild-type genomic background, some P. aeruginosa strains are unable to develop a type III dependent cytotoxicity except with an over-expression, in trans, of the transcriptional activator of this virulence system, ExsA. We proposed to call these strains non-inducible for type III cytotoxicity. This observation and the presence of a positive feedback loop regulating the expression of exsA led us to hypothesize the possibility of an epigenetic switch between inducibility/non inducibility of the type III dependant cytotoxicity in P. aeruginosa. With the help of generalised logic and a formal computer approach we designed a minimal model of P. aeruginosa type III basic regulation under calcium depletion and established the consistency of the hypothesis of an epigenetic acquisition of an inducible type III cytotoxicity. This model also showed that in this case, and in this case only, an artificial (exogenous) transient increase in protein ExsA would suffice to permanently switch a non inducible strain to inducibility. Next, we engineered an exsA inducible gene. With this construction, we demonstrated that a stable acquisition of the secretory phenotype after a transient signal relies on the presence of the feedback loop of the auto regulated exsA gene in agreement with the model. These non inducible strains thus switched to inducibility, also showed the acquisition of in vivo inducible type III cytotoxicity. Since the exsA gene added in trans was under the control of the inducible promoter, ptac, we have experienced leakiness or noise. This phenomenon, ubiquitous in biological systems, is responsible for cell-to-cell variation in gene expression. It has been shown to be the cause of population bimodal heterogeneity, when coupled with a feedback circuit responsible for bistability, for instance in the repartition of lytic/temperate phages in the population upon infection of E. coli by bacteriophage-λ . In some individuals in the population bearing pexsA ind such a noise-dependant switch could be responsible for the slight activity observed with non induced PAO1(pexsA ind) strain in in vitro experiments (Figure 4B, lane 3) and the in vivo increase of cytotoxicity of this strain. Moreover it has been shown in vitro that metabolic state, which is probably modified in lungs, regulates the percentage of cells able to induce type III secretion gene expression under inducing conditions evidencing that only a subpopulation could induce type III gene expression under inducing conditions. Although this does not prove that this is the mechanism involved to acquire or lose type III cytotoxicity during infection, the results obtained in vivo make the epigenetic hypothesis very likely. However, this hypothesis does not necessarily apply to the cytotoxicity of other bacteria, nor to mucoidy in P. aeruginosa for which an epigenetic acquisition had been proposed. In each case, the hypothesis must be tested, first by modelling then experimentally.
These in vitro and in vivo experiments, in accordance to the predictions of the formal method, indicate that a stable acquisition (or loss) of a phenotypic trait involved in the pathogenicity of P. aeruginosa can arise from an epigenetic switch. Direct therapeutic consequences could arise for P. aeruginosa infections especially in cystic fibrosis. Any therapy inhibiting the type III regulon would reduce the pathogenicity of the bacteria and the severity of the infection. Since the inducibility of type III secretory phenotype is a bistable phenotype, a reduction of ExsA activity below the triggering threshold would be sufficient to impede the positive feedback circuit leading to reversion of the type III secretory phenotype and reduction of the pathogenicity of the bacteria. To do so, we may investigate the use of quorum sensing signals as the Rhl quorum sensing system represses TTSS expression[30, 31] or the effect of small molecules able to block TTSS toxins secretion as shown in Yersinia which could lead to a repression of the system due to the absence of the secretion of the inhibitor ExsE. According to the hypothesis that planctonic type III secreting form of P. aeruginosa is responsible for early and invasive phases of the infection [33–35], such a therapy would improve antibiotic performance and other treatments to eradicate the infection or limit spreading.
Bacterial strains and growth conditions
Pseudomonas aeruginosa strains used in this study were: CHA, a cytotoxic isolate from a cystic fibrosis patient which secretes toxins under inducing condition for the type III secretion system; PAO1, a noncytotoxic strain widely used in laboratory studies; PAO1 ExsA-, an isogenic mutant of PAO1 unable to synthesize ExsA. PAO1 ExsA- was obtained using a mutation method based on sacB negative selection and cre-lox antibiotic marker recycling. Strains were routinely grown in Luria-Bertani broth (LB), Tryptic Soy Broth (TSB) or plated on PIA (Pseudomonas Isolation Agar, Difco, France). The pexsA ind plasmid was introduced into PAO1 and its isogenic mutant by electroporation and maintained with 300 μg of carbenicilin/ml.
Construction of an inducible exsA gene
PCR primers and probes used, restriction sites are in bold (6-FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine).
Amplification of exsA
Amplification of exsAind
GGGTACC TCACTGCCCGC TTTCCAGT
Reverse transcription of exsA
Amplification and detection of exsA cDNA in RT PCR
Amplification and detection of 16S rRNA cDNA in RT PCR
Total RNA were extracted after calcium depletion using HighPure RNA Extraction kit (Roche) according to the manufacturer's instructions. Reverse transcription was realized using the Transcriptor reverse transcriptase (Roche) with 1,33 μg total RNA, 1 mM dNTP and 1 mM of primer 16sas or RTExsA (Table 1). Primer and RNA were mixed, heated for 10 min at 65°C then dNTP, buffer and enzyme were added. cDNA were obtained after a reaction conducted for 30 min at 55°C, followed by 5 min at 85°C.
Real time PCR
Real-time PCR assays with hydrolysis probes were conducted on a LightCycler apparatus (Roche), using FastStart DNA Master Hybridization Probe kit (Roche) by following the manufacturer's instructions. Sequences of exsA gene and 16s rRNA gene used has housekeeping gene were obtained from the P. aeruginosa genome sequence from Pseudomonas Genome Project. Primers and probes (table 1) were designed and obtained from Proligo® (France) and were used at final concentrations of 0.75 μM and 0.2 μM, respectively. Final MgCl2 concentration was adjusted to 4 mM. Volume of cDNA from reverse transcription was 5 μl per assay. Thermal cycling conditions were 10 min at 95°C, followed by 45 cycles of 10 s at 95°C and 10 s at 58°C. Relative expression level of the exsA gene to the 16s rRNA gene was calculated using the Lightcycler software 4.0 for each strain and reported to exsA relative expression of the strain PAO1. Values are the mean (± standard error) of three experiments.
Bacteria were cultivated in LB broth overnight, diluted to 1.2 × 108cfu/ml in LB supplemented or not supplemented with 1 mM ITPG and incubated with aeration for 20 min at 37°C. Then, bacteria were spun down, washed twice in LB and grown with aeration for 3 h at 37°C in the presence or absence of 1 mM IPTG in conditions of calcium depletion: LB medium supplemented with 5 mM EGTA and 20 mM MgCl2 (inducing conditions known to induce secretion of type III toxins in vitro). Bacterial densities were determined by optical density measurement at 600 nm. Cultures were spun down, and proteins in the supernatant were precipitated by perchloric acid (precipitated volume was normalised to 9 × 108 cfu) and washed with acetone. Proteins were separated on a 12% SDS-PAGE, and stained with Coomassie blue. In the secretion experiment with a delay between the IPTG pulse and calcium depletion (Figure 4B), bacteria were pulsed with 1 mM ITPG for 20 min then washed with LB. Next, they were maintained in exponential phase of growth by serial dilution with fresh medium during a definite time before calcium depletion.
Animals and infection model
Specific pathogen-free Sprague Dawley rats (n = 40) (280–320 g), (Charles River Laboratoires France, St Germain/l'Arbresle, France) were housed in the Lille University Animal Care Facility and allowed food and water ad lib. All experiments were performed with approval of the Lille Institutional Animal Care and Use Committee. P. aeruginosa strains were cultivated in Triptic Soy Broth (TSB) broth overnight, diluted to 1.2 × 108cfu/ml in TSB supplemented or not supplemented with 1 mM ITPG and incubated with aeration for 20 min at 37°C. Cultures were then centrifuged, washed twice and grown with aeration at 37°C in tryptic soy broth medium. After 3 hours, bacteria were washed and resuspended in physiological serum to reach a final concentration of 108 or 2 × 109 cfu/ml evaluated by spectrophotometry. Acute lung injury was produced according to the method described by Pennington and Ehrie. Under short anaesthesia, a small midline incision was made on the neck ventral surface after swabbing it with ethanol. The trachea was exposed by blunt dissection. Using a 28-gauge needle, 0.5 ml/kg of bacterial suspension was instilled into the trachea, followed by injection of 0.5 ml of air[42, 43]. The animals were studied 24 h after instillation of the bacteria.
Bronchoalveolar lavage (BAL)
Bronchoalveolar lavage was performed by cannulating the trachea. Lungs from each experimental group were washed with a total of 20 ml in 5-ml aliquots of PBS with 3 mM EDTA. The returned fluid was pooled and centrifuged (200 g for 10 min). BAL fluid (BALF) samples were filtered and immediately frozen at -80°C after collection. Protein concentration in the BALF was measured with an automated analyzer (Hitachi 917, Japan).
This work was supported by a grant from the "association Vaincre la Mucoviscidose". We thank L. Quénée for technical assistance and Professors F. Morel and F. Molina, and the members of the epigenomic workshop in genopole for helpful discussions.
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