Synthetic gene-regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae

Streptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.</p

Transgenic mouse model harboring the transcriptional fusion ccl20-luciferase as a novel reporter of pro-inflammatory response

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.Fil: Crispo, Martina. Unidad de Animales Transgénicos y de Experimentación – Institut Pasteur de Montevideo; UruguayFil: Van Maele, Laurye. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Tabareau, Julien. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Cayet, Delphine. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Errea, Agustina Juliana. Universidad Nacional de la Plata. Facultad de Cs.exactas. Departamento de Cs.biologicas. Laboratorio de Invest.del Sistema Inmune; ArgentinaFil: Ferreira, Ana María. Cátedra de Inmunologíaa. Facultad de Ciencias/Facultad de Química. Universidad de la República; UruguayFil: Rumbo, Martin. Universidad Nacional de la Plata. Facultad de Cs.exactas. Departamento de Cs.biologicas. Laboratorio de Invest.del Sistema Inmune; ArgentinaFil: Sirard, Jean Claude. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; Franci

Airway structural cells regulate TLR5-mediated mucosal adjuvant activity

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin’s mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.Fil: Van Maele, Laurye. Institut Pasteur de Lille. Lille; Francia. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Fougeron, Delphine. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Janot, Laurent. University of Orléans. Orléans; Francia. Institut de Transgenose. Orleans; FranciaFil: Didierlaurent, A.. Imperial College of London. Londres; Reino UnidoFil: Cayet, D.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Tabareau, J.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Corvo Chamaillard, S.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Boulenoir, S.. Institut Pasteur de Lille. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Univ Lille Nord de France. Lille; FranciaFil: Jeffs, S. Imperial College of London. Londres; Reino UnidoFil: Vande Walle, L. Department of Medical Protein Research. Ghent; Bélgica. University of Ghent; BélgicaFil: Lamkanfi, M.. Department of Medical Protein Research. Ghent; Bélgica. University of Ghent; BélgicaFil: Lemoine, Y.. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Institut Pasteur de Lille. Lille; FranciaFil: Erard, F.. Institut de Transgenose. Orleans; Francia. University of Orléans. Orléans; FranciaFil: Hot, D.. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Institut Pasteur de Lille. Lille; FranciaFil: Hussell, Tracy. Imperial College of London. Londres; Reino Unido. University of Manchester; Reino UnidoFil: Ryffel, B.. Institut de Transgenose. Orleans; Francia. University of Orléans. Orléans; FranciaFil: Benecke, Arndt G.. Institut des Hautes Études Scientifiques and Centre National de la Recherche Scientifique; FranciaFil: Sirard, J.C.. Univ Lille Nord de France. Lille; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Institut Pasteur de Lille. Lille; Franci

IL-17A/F-Signaling Does Not Contribute to the Initial Phase of Mucosal Inflammation Triggered by S. Typhimurium

Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium) causes diarrhea and acute inflammation of the intestinal mucosa. The pro-inflammatory cytokines IL-17A and IL-17F are strongly induced in the infected mucosa but their contribution in driving the tissue inflammation is not understood. We have used the streptomycin mouse model to analyze the role of IL-17A and IL-17F and their cognate receptor IL-17RA in S. Typhimurium enterocolitis. Neutralization of IL-17A and IL-17F did not affect mucosal inflammation triggered by infection or spread of S. Typhimurium to systemic sites by 48 h p.i. Similarly, Il17ra−/− mice did not display any reduction in infection or inflammation by 12 h p.i. The same results were obtained using S. Typhimurium variants infecting via the TTSS1 type III secretion system, the TTSS1 effector SipA or the TTSS1 effector SopE. Moreover, the expression pattern of 45 genes encoding chemokines/cytokines (including CXCL1, CXCL2, IL-17A, IL-17F, IL-1α, IL-1β, IFNγ, CXCL-10, CXCL-9, IL-6, CCL3, CCL4) and antibacterial molecules was not affected by Il17ra deficiency by 12 h p.i. Thus, in spite of the strong increase in Il17a/Il17f mRNA in the infected mucosa, IL-17RA signaling seems to be dispensable for eliciting the acute disease. Future work will have to address whether this is attributable to redundancy in the cytokine signaling network

The Microbiota Mediates Pathogen Clearance from the Gut Lumen after Non-Typhoidal Salmonella Diarrhea

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tmatt, sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRβ−/−δ−/−, JH−/−, IgA−/−, pIgR−/−). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using ‘L-mice’ which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tmatt from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most “classical” immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission

Propriétés immuno-modulatrices de la flagelline de Salmonella typhimurium : impact sur la défense anti-bactérienne et le développement d'adjuvants épithéliaux

The mammalian immune system has two arms, the innate immunity, first line of defense, and the adaptive immunity, operating later and antigen-specific. During infection, immune responses begin with the detection of microbes by innate receptors such as the Toll-Like Receptors (TLR). These receptors detect molecular motifs highly conserved by microorganisms like lipopolysaccharide or flagellin. As most pathogens infect mammals by respiratory, digestive, urogenital tracts, these specialized barriers are thus essential sites for anti-microbial immunity. In this work, I specifically studied, the Salmonella typhimurium flagellin, structural molecule of bacterial flagellum and TLR5 agonist. My work aims at dissecting mucosal immunes responses induced by the TLR5 signalisation by two experimental models : (1) a local administration mimicking microbial colonization of mucosa and (2) a systemic administration mimicking pathogen invasion in mucosal tissues. Flagellin administration into the respiratory tract activated immune responses. Previous studies demonstrated that flagellin-mediated innate responses are controlled by epithelial cells and the stimulation was sufficient to chemokine and cytokine secretion. This work showed that the TLR5-mediated epithelial response promoted neutrophil-dependent clearance of Streptococcus pneumoniae in a model of respiratory infection. Besides, the role of epithelial cells of the broncho-alveolar mucosa in TLR-dependent initiation of adaptive immunity was analyzed. Upon nasal immunization with flagellin as adjuvant, animals developed a local and systemic antigen-specific antibody and mixed Th1 and Th2 responses. Our results suggested that lung epithelial cells are necessary to promote the mucosal and systemic immunity. These observations open unique perspectives to develop mucosal adjuvants and therapies with an epithelial activity and to characterize host/pathogen interactions. The systemic administration of flagellin induced in mucosa a swift and transient transcription of genes encoding the interleukins IL-17A, IL-17F and IL-22, cytokines crucial to elaborate a mucosal antimicrobial defense. The source of the Th17-related cytokines was identified as a novel population of CD3negCD127+ innate lymphoid cells. Their activation is associated to protection against oral infection with Yersinia pseudotuberculosis. Therefore, these data suggest that activation of CD3negCD127+ cells is instrumental for the early defenses against pathogen invasion of host mucosal tissues. Intervention on these innate lymphoid cells may represent a novel antimicrobial approach.Le système immunitaire des mammifères possède deux composantes, l'immunité innée, première ligne de défense de l'hôte, et l'immunité adaptative, phase plus tardive et spécifique à de multiples antigènes. Dans le contexte infectieux, toute réponse immunitaire commence par la détection du pathogène par des récepteurs de l'immunité innée comme les "Toll-Like Receptors" ou TLR qui reconnaissent des motifs moléculaires conservés chez de nombreux microorganismes. Au cours de ce travail, j'ai étudié un motif microbien en particulier, la flagelline de Salmonella typhimurium, molécule structurale du flagelle et agoniste de TLR5. Les muqueuses respiratoires, digestives ou urogénitales sont donc des sites majeurs d'immunité anti-microbienne car la plupart des pathogènes les ciblent et les colonisent. Mon travail a consisté à disséquer les réponses immunitaires muqueuses induites par la signalisation TLR5 dans deux modèles expérimentaux : (1) une administration locale de la flagelline mimant la colonisation microbienne épithéliale et (2) une administration systémique simulant l'invasion d'un pathogène dans les tissus muqueux. L'instillation de flagelline par voie respiratoire active l'épithélium broncho-alvéolaire. Nos travaux ont montré que la signalisation épithéliale peut avoir un effet thérapeutique par le recrutement de neutrophiles. La contribution de l'épithélium dans l'immunité adaptative muqueuse a été analysée. Lors d'une instillation nasale de flagelline, les animaux développent une réponse en anticorps et une réponse Th1-Th2 à la fois locale et systémique. Nos résultats indiquent que la signalisation TLR dans les cellules épithéliales du poumon est nécessaire et suffisante pour promouvoir cette immunité. Ces observations ouvrent de nouvelles perspectives pour le développement d'adjuvant muqueux à activité épithéliale et la caractérisation des interactions hôte-pathogène. L'administration systémique de flagelline induit dans l'intestin et les poumons la transcription immédiate mais transitoire des gènes codant les interleukines IL-17A, IL-17F et IL-22, cytokines essentielles à l'élaboration d'une défense anti-microbienne muqueuse. Nous avons identifié comme source de ces cytokines, une nouvelle population de cellules lymphoïdes innées CD3negCD127+. Leur activation est associée à la protection contre une infection orale à Yersinia pseudotuberculosis. Nos données suggèrent donc que les cellules CD3negCD127+ sont cruciales pour les défenses précoces contre l'invasion des tissus muqueux par les pathogènes et leur manipulation pourrait être envisagées à des fins thérapeutiques

Propriétés immuno-modulatrices de la flagelline de Salmonella typhimurium (impact sur la défense anti-bactérienne et le développement d'adjuvants épithéliaux)

Le système immunitaire des mammifères possède deux composantes, l'immunité innée, première ligne de défense de l'hôte, et l'immunité adaptative, phase plus tardive et spécifique à de multiples antigènes. Dans le contexte infectieux, toute réponse immunitaire commence par la détection du pathogène par des récepteurs de l'immunité innée comme les "Toll-Like Receptors" ou TLR qui reconnaissent des motifs moléculaires conservés chez de nombreux microorganismes. Au cours de ce travail, j'ai étudié un motif microbien en particulier, la flagelline de Salmonella typhimurium, molécule structurale du flagelle et agoniste de TLR5. Les muqueuses respiratoires, digestives ou urogénitales sont donc des sites majeurs d'immunité anti-microbienne car la plupart des pathogènes les ciblent et les colonisent. Mon travail a consisté à disséquer les réponses immunitaires muqueuses induites par la signalisation TLR5 dans deux modèles expérimentaux : (1) une administration locale de la flagelline mimant la colonisation microbienne épithéliale et (2) une administration systémique simulant l'invasion d'un pathogène dans les tissus muqueux. L'instillation de flagelline par voie respiratoire active l'épithélium broncho-alvéolaire. Nos travaux ont montré que la signalisation épithéliale peut avoir un effet thérapeutique par le recrutement de neutrophiles. La contribution de l'épithélium dans l'immunité adaptative muqueuse a été analysée. Lors d'une instillation nasale de flagelline, les animaux développent une réponse en anticorps et une réponse Th1-Th2 à la fois locale et systémique. Nos résultats indiquent que la signalisation TLR dans les cellules épithéliales du poumon est nécessaire et suffisante pour promouvoir cette immunité. Ces observations ouvrent de nouvelles perspectives pour le développement d'adjuvant muqueux à activité épithéliale et la caractérisation des interactions hôte-pathogène. L'administration systémique de flagelline induit dans l'intestin et les poumons la transcription immédiate mais transitoire des gènes codant les interleukines IL-17A, IL-17F et IL-22, cytokines essentielles à l'élaboration d'une défense anti-microbienne muqueuse. Nous avons identifié comme source de ces cytokines, une nouvelle population de cellules lymphoïdes innées CD3negCD127+. Leur activation est associée à la protection contre une infection orale à Yersinia pseudotuberculosis. Nos données suggèrent donc que les cellules CD3negCD127+ sont cruciales pour les défenses précoces contre l'invasion des tissus muqueux par les pathogènes et leur manipulation pourrait être envisagées à des fins thérapeutiques.The mammalian immune system has two arms, the innate immunity, first line of defense, and the adaptive immunity, operating later and antigen-specific. During infection, immune responses begin with the detection of microbes by innate receptors such as the Toll-Like Receptors (TLR). These receptors detect molecular motifs highly conserved by microorganisms like lipopolysaccharide or flagellin. As most pathogens infect mammals by respiratory, digestive, urogenital tracts, these specialized barriers are thus essential sites for anti-microbial immunity. In this work, I specifically studied, the Salmonella typhimurium flagellin, structural molecule of bacterial flagellum and TLR5 agonist. My work aims at dissecting mucosal immunes responses induced by the TLR5 signalisation by two experimental models : (1) a local administration mimicking microbial colonization of mucosa and (2) a systemic administration mimicking pathogen invasion in mucosal tissues. Flagellin administration into the respiratory tract activated immune responses. Previous studies demonstrated that flagellin-mediated innate responses are controlled by epithelial cells and the stimulation was sufficient to chemokine and cytokine secretion. This work showed that the TLR5-mediated epithelial response promoted neutrophil-dependent clearance of Streptococcus pneumoniae in a model of respiratory infection. Besides, the role of epithelial cells of the broncho-alveolar mucosa in TLR-dependent initiation of adaptive immunity was analyzed. Upon nasal immunization with flagellin as adjuvant, animals developed a local and systemic antigen-specific antibody and mixed Th1 and Th2 responses. Our results suggested that lung epithelial cells are necessary to promote the mucosal and systemic immunity. These observations open unique perspectives to develop mucosal adjuvants and therapies with an epithelial activity and to characterize host/pathogen interactions. The systemic administration of flagellin induced in mucosa a swift and transient transcription of genes encoding the interleukins IL-17A, IL-17F and IL-22, cytokines crucial to elaborate a mucosal antimicrobial defense. The source of the Th17-related cytokines was identified as a novel population of CD3negCD127+ innate lymphoid cells. Their activation is associated to protection against oral infection with Yersinia pseudotuberculosis. Therefore, these data suggest that activation of CD3negCD127+ cells is instrumental for the early defenses against pathogen invasion of host mucosal tissues. Intervention on these innate lymphoid cells may represent a novel antimicrobial approach.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

The GM-CSF released by airway epithelial cells orchestrates the mucosal adjuvant activity of flagellin

International audienceThe TLR5 agonist flagellin is a potent adjuvant and is currently being developed for use in vaccines. The mechanisms that drive flagellin's activity are influenced by its administration route. Previous studies showed that lung structural cells (especially epithelial cells lining the conducting airways) are pivotal for the efficacy of intranasally administered flagellin-containing vaccines. In this study, we looked at how the airway epithelial cells (AECs) regulate the flagellin-dependent stimulation of Ag-specific CD4+ T cells and the Ab response in mice. Our results demonstrate that after sensing flagellin, AECs trigger the release of GM-CSF in a TLR5-dependent fashion and the doubling of the number of activated type 2 conventional dendritic cells (cDC2s) in draining lymph nodes. Furthermore, the neutralization of GM-CSF reduced cDC2s activation. This resulted in lower of Ag-specific CD4+ T cell count and Ab titers in mice. Our data indicate that during pulmonary immunization, the GM-CSF released by AECs orchestrates the cross-talk between cDC2s and CD4+ T cells and thus drives flagellin's adjuvant effect

Mucosal Administration of Flagellin Protects Mice from Streptococcus pneumoniae Lung Infection▿

Streptococcus pneumoniae is a major cause of pneumonia in infants and the elderly. Innate defenses are essential to the control of pneumococcal infections, and deficient responses can trigger disease in susceptible individuals. Here we showed that flagellin can locally activate innate immunity and thereby increase the resistance to acute pneumonia. Flagellin mucosal treatment improved S. pneumoniae clearance in the lungs and promoted increased survival of infection. In addition, lung architecture was fully restored after the treatment of infected mice, indicating that flagellin allows the reestablishment of steady-state conditions. Using a flagellin mutant that is unable to signal through Toll-like receptor 5 (TLR5), we established that TLR5 signaling is essential for protection. In the respiratory tract, flagellin induced neutrophil infiltration into airways and upregulated the expression of genes coding for interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, and CCL20. Using depleting antibodies, we demonstrated that neutrophils are major effectors of protection. Further, we found that B- and T-cell-deficient SCID mice clear S. pneumoniae challenge to the same extent as immunocompetent animals, suggesting that these cell populations are not required for flagellin-induced protection. In conclusion, this study emphasizes that mucosal stimulation of innate immunity by a TLR not naturally engaged by S. pneumoniae can increase the potential to cure pneumococcal pneumonia

Toll‐like receptor 4 signaling in hematopoietic‐lineage cells contributes to the enhanced activity of the human vaccine adjuvant AS01

International audienceThe 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01