Epigenetic acquisition of inducibility of type III cytotoxicity in P. aeruginosa

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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

MECANISMES DE REGULATION IMPLIQUES DANS LA PATHOGENICITE DE PSEUDOMONAS AERUGINOSA : SYSTEME DE SECRETION DE TYPE III, EPIGENESE ET QUORUM SENSING

Pseudomonas aeruginosa is an opportunistic bacterium responsible for severe infections. Its pathogenicity comes from many virulence factors including the type III secretion system (TTSS). This system is activated by contact between the cell and the bacterium or by calcium depletion of the medium and allows injection of toxins into host cell cytosol. Different phenotypes are observed in chronic lung infection: an inducible phenotype by cell contact or calcium depletion and a non inducible one.If no mutations have occurred, those two phenotypes could be explained by epigenesis.With the help of a bioinformatic tool, we established all possible dynamics of the minimal TTSS regulatory model which fit the bistability hypothesis and showed the possibility of epigenesis. Through this method, we also design descriminating experiments to validate the hypothesis and first demonstrated the epigenetic acquisition of the capacity for the type III secretion system of P. aeruginosa to be activated by calcium depletion. The possibility of this epigenetic phenotypic switch was then confirmed in vivo in an acute pulmonary infection model.On an other hand, we showed that TTSS is repressed in high cell density condition. This repression is induced by a signal produced and secreted by P. aeruginosa. Using mutants we demonstrated that known quorum sensing signals are not responsible for this inhibition. Thus, TTSS expression is cell density dependent and is repressed at high cell density by an unknown quorum sensing like mechanism.Pseudomonas aeruginosa est un bacille opportuniste responsable d'infections graves. Sa pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT). Ce système est activé par le contact de la bactérie avec une cellule ou une déplétion calcique et permet l'injection de toxines directement dans le cytosol de la cellule. Différents phénotypes sont observés lors d'une infection pulmonaire dans le cas de la mucoviscidose : un phénotype inductible par le contact cellulaire ou la déplétion calcique et un autre non inductible.En l'absence de mutations, cette dualité de phénotype peut être envisagée sous un aspect épigénétique.A l'aide d'un outil informatique, nous avons déterminé les dynamiques possibles d'un modèle du SSTT supportant l'hypothèse de bistabilité et mis en évidence l'existence possible d'épigénèse. Grâce à cette méthode nous avons également définit les expériences permettant de tester cette hypothèse. Nous avons démontré qu'une modification épigénétique pouvait être à l'origine d'une acquisition stable de l'inductibilité du SSTT in vitro. Ce changement héréditaire de phénotype a été confirmé, in vivo, à l'aide d'un modèle d'infection pulmonaire aiguë.Dans un second temps, nous avons mis en évidence une répression du SSTT à densité cellulaire élevée. Celle-ci est induite par un signal produit et sécrété par la bactérie. L'utilisation de mutants a permis de montrer que les signaux connus du quorum sensing ne sont pas impliqués dans cette répression. Ainsi, l'expression du SSTT dépend de la densité bactérienne et la répression à densité cellulaire élevée est induite par un mécanisme de type quorum sensing non connu

Mécanismes de régulation impliqués dans la pathogénicité de Pseudomonas aeruginosa (système de sécrétion de type III, épigénèse et quorum sensing)

Pseudomonas aeruginosa est un bacille opportuniste responsable d'infections graves. Sa pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT). Ce système est activé par le contact de la bactérie avec une cellule ou une déplétion calcique et permet l'injection de toxines directement dans le cytosol de la cellule. Différents phénotypes sont observés lors d'une infection pulmonaire dans le cas de la mucoviscidose : un phénotype inductible par le contact cellulaire ou la déplétion calcique et un autre non inductible. En l'absence de mutations, cette dualité de phénotype peut être envisagée sous un aspect épigénétique. A l'aide d'un outil informatique, nous avons déterminé les dynamiques possibles d'un modèle du SSTT supportant l'hypothèse de bistabilité et mis en évidence l'existence possible d'épigénèse. Grâce à cette méthode nous avons également définit les expériences permettant de tester cette hypothèse. Nous avons démontré qu'une modification épigénétique pouvait être à l'origine d'une acquisition stable de l'inductibilité du SSTT in vitro. Ce changement héréditaire de phénotype a été confirmé, in vivo, à l'aide d'un modèle d'infection pulmonaire aiguë. Dans un second temps, nous avons mis en évidence une répression du SSTT à densité cellulaire élevée. Celle-ci est induite par un signal produit et sécrété par la bactérie. L'utilisation de mutants a permis de montrer que les signaux connus du quorum sensing ne sont pas impliqués dans cette répression. Ainsi, l'expression du SSTT dépend de la densité bactérienne et la répression à densité cellulaire élevée est induite par un mécanisme de type quorum sensing non connu.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Optimization of a Type III Secretion System-Based Pseudomonas aeruginosa Live Vector for Antigen Delivery▿

During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. We showed that a type III secretion-based Pseudomonas aeruginosa vector delivering the ovalbumin (OVA) antigen induced an efficient specific CD8+ T-lymphocyte immune response against OVA-expressing cells. Because of the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we explored the effects of the deletion of quorum-sensing genes and the aroA gene toward toxicity and efficiency of the vector strain. The aroA mutation in our strain (making the strain auxotrophic for aromatic amino acids) conferred a strikingly reduced toxicity, with the bacterial lethal dose being more than 100 times higher than that of the parental strain. The quorum-sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, an OVA-delivering aroA-deficient mutant was the most efficient at a low dose (105), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (106), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (107), in agreement with the known negative effects of quorum-sensing molecules upon T-cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding toxicity and efficiency for further developments in both antitumor and anti-infectious strategies

High-cell-density regulation of the Pseudomonas aeruginosa type III secretion system: implications for tryptophan catabolites.

International audienceThe Pseudomonas aeruginosa type III secretion system (T3SS) is known to be a very important virulence factor in acute human infections, but it is less important in maintaining chronic infections in which T3SS genes are downregulated. In vitro, the activation of T3SS expression involves a positive activating loop that acts on the transcriptional regulator ExsA. We have observed that in vivo T3SS expression is cell density-dependent in a manner that does not need known quorum-sensing (QS) signals. In addition, stationary-phase culture supernatants added to exponential-phase growing strains can inhibit T3SS expression. The analysis of transposon insertion mutants showed that the production of such T3SS-inhibiting signals might depend on tryptophan synthase and hence tryptophan, which is the precursor of signalling molecules such as indole-3-acetic acid (IAA), kynurenine and Pseudomonas quinolone signal (PQS). Commercially available tryptophan-derived molecules were tested for their role in the regulation of T3SS expression. At millimolar concentrations, IAA, 1-naphthalacetic acid (NAA) and 3-hydroxykynurenine inhibited T3SS expression. Inactivation of the tryptophan dioxygenase-encoding kynA gene resulted in a decrease in the T3SS-inhibiting activity of supernatants. These observations suggest that tryptophan catabolites are involved in the downregulation of T3SS expression in the transition from a low- to a high-cell-density state

Anti-tumor immunotherapy via antigen delivery from a live attenuated genetically engineered Pseudomonas aeruginosa type III secretion system-based vector.

Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigen-presenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8(+) T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8(+) T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy

A Legionella pneumophila effector impedes host gene silencing to promote virulence

Posted November 17, 2022 on bioRxiv.RNA silencing is a gene silencing mechanism directed by siRNAs and miRNAs. Human miRNAs act as central regulators of host-bacteria interactions. However, it is unknown whether human pathogenic bacteria could impede RNA silencing to promote virulence. Here, we show that the Legionella pneumophila type IV-secreted effector LegK1 suppresses siRNA- and miRNA-activities in human cells. This ability depends on its kinase activity and on a functional tryptophan-dependent Argonaute (Ago)-binding platform. We further show that the capacity of LegK1 to activate NF- κ B signaling contributes to silencing suppression, demonstrating a link between effector-mediated NF- κ B signaling activation and silencing suppression. LegK1 also promotes L. pneumophila growth in both amoeba and human macrophages, supporting a key role of this effector in virulence. In infected macrophages, the latter activity relies on the genetic targeting of human Ago4, highlighting a novel function of this host factor in antibacterial resistance