Abstract
BackgroundPseudomonas 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.ResultsUsing 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.ConclusionThese results may offer new perspectives for therapeutic strategies to prevent lethal infections by P. aeruginosa by reverting the epigenetic inducibility of type III cytotoxicity.
Highlights
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
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
The hypothesis must be tested, first by modelling experimentally. These in vitro and in vivo experiments, in accordance to the predictions of the formal method, indicate that a stable acquisition of a phenotypic trait involved in the pathogenicity of P. aeruginosa can arise from an epigenetic switch
Summary
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. Cytotoxic P. aeruginosa inject toxins from their cytoplasm into eukaryotic target cells through a protein secretory apparatus, the type III secretion system (TTSS)[2] Activation of this system (especially toxins production and secretion) is dependant on the contact between the bacteria and the host cells in vivo and could be triggered by calcium depletion of the growth medium in vitro. Upon TTSS activation by calcium depletion, ExsE is secreted through it, releasing ExsC and the inhibition of ExsA by ExsD[6,7] This complex regulatory network is described in figure 1A. Numerous strains possessing the entire set of genes required for type III cytotoxicity cannot be induced by host contact or calcium depletion unless submitted to exsA overexpression[8,9]. It has recently been shown that they accumulate in the lungs of cystic fibrosis patients during long term infection
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