Boosting the IL-22 response using flagellin prevents bacterial infection in cigarette smoke-exposed mice.

The progression of chronic obstructive pulmonary disease (COPD), a lung inflammatory disease being the fourth cause of death worldwide, is marked by acute exacerbations. These episodes are mainly caused by bacterial infections, frequently due to Streptococcus pneumoniae. This susceptibility to infection involves a defect in interleukin (IL)-22, which plays a pivotal role in mucosal defense mechanism. Administration of flagellin, a Toll-like receptor 5 (TLR-5) agonist, can protect mice and primates against respiratory infections in a non-pathological background. We hypothesized that TLR-5-mediated stimulation of innate immunity might improve the development of bacteria-induced exacerbations in a COPD context. Mice chronically exposed to cigarette smoke (CS), mimicking COPD symptoms, are infected with S. pneumoniae, and treated in a preventive and a delayed manner with flagellin. Both treatments induced a lower bacterial load in the lungs and blood, and strongly reduced the inflammation and lung lesions associated with the infection. This protection implicated an enhanced production of IL-22 and involved the recirculation of soluble factors secreted by spleen cells. This is also associated with higher levels of the S100A8 anti-microbial peptide in the lung. Furthermore, human mononuclear cells from non-smokers were able to respond to recombinant flagellin by increasing IL-22 production while active smoker cells do not, a defect associated with an altered IL-23 production. This study shows that stimulation of innate immunity by a TLR-5 ligand reduces CS-induced susceptibility to bacterial infection in mice, and should be considered in therapeutic strategies against COPD exacerbations.

mTOR-driven glycolysis governs induction of innate immune responses by bronchial epithelial cells exposed to the bacterial component flagellin.

Human bronchial epithelial (HBE) cells play an essential role during bacterial infections of the airways by sensing pathogens and orchestrating protective immune responses. We here sought to determine which metabolic pathways are utilized by HBE cells to mount innate immune responses upon exposure to a relevant bacterial agonist. Stimulation of HBE cells by the bacterial component flagellin triggered activation of the mTOR pathway resulting in an increased glycolytic flux that sustained the secretory activity of immune mediators by HBE cells. The mTOR inhibitor rapamycin impeded glycolysis and limited flagellin-induced secretion of immune mediators. The role of the mTOR pathway was recapitulated in vivo in a mouse model of flagellin-triggered lung innate immune responses. These data demonstrate that metabolic reprogramming via the mTOR pathway modulates activation of the respiratory epithelium, identifying mTOR as a potential therapeutic target to modulate mucosal immunity in the context of bacterial infections.

Toll-like receptor 2 is critical for induction of Reg3 beta expression and intestinal clearance of Yersinia pseudotuberculosis.

OBJECTIVE: Yersinia pseudotuberculosis causes ileitis and mesenteric lymphadenitis by mainly invading the Peyer's patches that are positioned in the terminal ileum. Whereas toll-like-receptor 2 (TLR2) controls mucosal inflammation by detecting certain microbiota-derived signals, its exact role in protecting Peyer's patches against bacterial invasion has not been defined. DESIGN: Wild-type, Tlr2-, Nod2- and MyD88-deficient animals were challenged by Y pseudotuberculosis via the oral or systemic route. The role of microbiota in conditioning Peyer's patches against Yersinia through TLR2 was assessed by delivering, ad libitum, exogenous TLR2 agonists in drinking water to germ-free and streptomycin-treated animals. Bacterial eradication from Peyer's patches was measured by using a colony-forming unit assay. Expression of cryptdins and the c-type lectin Reg3 beta was quantified by quantitative reverse transcriptase polymerase chain reaction analysis. RESULTS: Our data demonstrated that Tlr2-deficient mice failed to limit Yersinia dissemination from the Peyer's patches and succumbed to sepsis independently of nucleotide-binding and oligomerisation domain 2 (NOD2). Recognition of both microbiota-derived and myeloid differentiation factor 88 (MyD88)-mediated elicitors was found to be critically involved in gut protection against Yersinia-induced lethality, while TLR2 was dispensable to systemic Yersinia infection. Gene expression analyses revealed that optimal epithelial transcript level of the anti-infective Reg3 beta requires TLR2 activation. Consistently, Yersinia infection triggered TLR2-dependent Reg3 beta expression in Peyer's patches. Importantly, oral treatment with exogenous TLR2 agonists in germ-free animals was able to further enhance Yersinia-induced expression of Reg3 beta and to restore intestinal resistance to Yersinia. Lastly, genetic ablation of Reg3 beta resulted in impaired clearance of the bacterial load in Peyer's patches. CONCLUSIONS: TLR2/REG3 beta is thus an essential component in conditioning epithelial defence signalling pathways against bacterial invasion.

Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella.

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responses.

Neutrophilic NLRP3 inflammasome-dependent IL-1ß secretion regulates the γδT17 cell response in respiratory bacterial infections.

Traditionally regarded as simple foot soldiers of the innate immune response limited to the eradication of pathogens, neutrophils recently emerged as more complex cells endowed with a set of immunoregulatory functions. Using a model of invasive pneumococcal disease, we highlighted an unexpected key role for neutrophils as accessory cells in innate interleukin (IL)-17A production by lung resident Vγ6Vδ1+ T cells via nucleotide-binding oligomerization domain receptor, pyrin-containing 3 (NLRP3) inflammasome-dependent IL-1ß secretion. In vivo activation of the NLRP3 inflammasome in neutrophils required both host-derived and bacterial-derived signals. Elaborately, it relies on (i) alveolar macrophage-secreted TNF-α for priming and (ii) subsequent exposure to bacterial pneumolysin for activation. Interestingly, this mechanism can be translated to human neutrophils. Our work revealed the cellular and molecular dynamic events leading to γδT17 cell activation, and highlighted for the first time the existence of a fully functional NLRP3 inflammasome in lung neutrophils. This immune axis thus regulates the development of a protective host response to respiratory bacterial infections.

Molecular tools for the study of transcriptional regulation in Bacillus anthracis.

Bacillus anthracis produces two toxins composed of three proteins. Genetic tools were constructed to study the regulation of toxin synthesis. They included transcriptional fusions with various reporter genes, in replicative and integrative vectors. The reporter gene xylE, encoding catechol 2,3-dioxygenase, may be valuable for screening of strong promoters, as expression of the gene can be visualized directly and the studies of regulation in B. anthracis. Therefore, transcriptional fusions between a lacZ reporter gene and the toxin genes were constructed. Experiments with a multicopy plasmid in trans suggested that the transcriptional activator(s) of the toxin genes were not titrated. B. anthracis strains, which contain pXO1 carrying multiple copies of fusions, were analysed. Expression of the reporter gene was proportional to the fusion copy number. Indeed, single integration of a suicide plasmid can be distinguished from multiple integration according to the level of resistance to an appropriate antibiotic. Finally, recombination in B. anthracis was found to be very efficient (approximately 10(-2) recombinants per transconjugant cell.

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.

The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature.

The two Bacillus anthracis toxins are composed of three proteins, protective antigen, lethal factor, and edema factor. The structural genes for these three components are located on the virulence plasmid pXO1. We constructed transcriptional fusions between the regulatory region of each of these genes and lacZ. Each construct was then inserted as a single copy at the corresponding toxin gene locus on pXO1, resulting in three isogenic strains. Two environmental factors, bicarbonate and temperature, were found to induce beta-galactosidase synthesis in each recombinant strain. Furthermore, the transcription of the three toxin genes appears to be coordinately regulated.

Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase.

The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N-terminal two-thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I. The suggested active-site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.

Antigen delivery by attenuated Bacillus anthracis: new prospects in veterinary vaccines.

This report summarizes the recent investigations on the use of Bacillus anthracis as a live vector for delivery of antigens. Recombinant strains were constructed by engineering the current live Sterne vaccine. This vaccine, used to prevent anthrax in cattle, causes side-effects due to anthrax toxin activities. Bacteria producing a genetically detoxified toxin factor were devoid of lethal effects and were as protective as the Sterne strain against experimental anthrax. Moreover, B. anthracis expressing a foreign antigen controlled by an in vivo inducible promoter were able to generate either antibody or cellular protective responses against heterologous diseases.

Live attenuated Salmonella: a paradigm of mucosal vaccines.

Two key steps control immune responses in mucosal tissues: the sampling and transepithelial transport of antigens, and their targeting into professional antigen-presenting cells in mucosa-associated lymphoid tissue. Live Salmonella bacteria use strategies that allow them to cross the epithelial barrier of the gut, to survive in antigen-presenting cells where bacterial antigens are processed and presented to the immune cells, and to express adjuvant activity that prevents induction of oral tolerance. Two Salmonella serovars have been used as vaccines or vectors, S. typhimurium in mice and S. typhi in humans. S. typhimurium causes gastroenteritis in a broad host range, including humans, while S. typhi infection is restricted to humans. Attenuated S. typhimurium has been used successfully in mice to induce systemic and mucosal responses against more than 60 heterologous antigens. This review aims to revisit S. typhimurium-based vaccination, as an alternative to S. typhi, with special emphasis on the molecular pathogenesis of S. typhimurium and the host response. We then discuss how such knowledge constitutes the basis for the rational design of novel live mucosal vaccines.

Protective antigen-mediated antibody response against a heterologous protein produced in vivo by Bacillus anthracis.

Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracis strains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinant B. anthracis strains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.

Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea.

Rotavirus is the major cause of diarrhea among young infants in both humans and animals. Immune protection of newborns by vaccination is difficult to achieve since there is not enough time to mount an immune response before exposure to the virus. We have designed a vaccination strategy mediating transfer of neutralizing antibodies from the mother to the offspring during pregnancy and/or lactation. Adult female mice were nasally immunized with virus-like particles (VLPs) made of viral proteins VP2 and 6 (VLP2/6) or VP 2, 6, and 7 (VLP2/6/7) derived from the RF rotavirus strain in the presence or absence of cholera toxin. Both vaccines elicited serum and milk antibodies against the respective VPs. Four days after parturition, suckling pups were challenged orally with RF rotavirus. Pups from mothers immunized with VLP2/6/7 but not VLP2/6 were protected against rotavirus diarrhea, indicating that VP7 plays a key role in protection. Protection was mediated by milk rather than serum antibodies, and mucosal adjuvants were not required. In conclusion, VLPs containing VP7 administered nasally to mothers represent a promising vaccine candidate for the protection of suckling newborns against rotavirus-induced diarrhea, even in the absence of a mucosal adjuvant.

Characterization of a plasmid region involved in Bacillus anthracis toxin production and pathogenesis.

The germination of spores within the host is the initial step of anthrax infection. We have shown, using immunofluorescence staining, confocal scanning laser microscopy and image cytometry analysis, that the alveolar macrophage is the primary site of B. anthracis germination in a murine inhalation infection model. B. anthracis germinated inside macrophages, in vesicles derived from the phagosomal compartment. We have demonstrated that the toxin genes and their trans-activator, AtxA, are expressed within the macrophages after germination. It was also shown that the pXO1 plasmid strongly enhanced capsule formation and that this influence is mediated by AtxA. This indicates the existence of a regulon where AtxA is the regulatory protein acting on genes located on different plasmids. We identified a tricistronic germination operon gerX located between the pag and atxA genes on the 40-kb toxin-encoding fragment of pXO1 . Analysis of a gerX null mutant indicated that gerX-encoded proteins are involved in the virulence of B. anthracis.

Entry and survival of Salmonella typhimurium in dendritic cells and presentation of recombinant antigens do not require macrophage-specific virulence factors.

Macrophages have long been regarded as the main target encountered by Salmonella typhimurium, a Gram-negative facultative intracellular pathogen that invades the intestinal mucosa. S. typhimurium, however, are first internalized by dendritic cells. To gain new insights into the interactions between Salmonella and the dendritic cells, we compared the fate of wild-type S. typhimurium and the virulence-attenuated PhoP constitutive (PhoP(c)) strain. The PhoP(c) strain is impaired for entry and survival in mammalian cells and is poorly processed by macrophages for antigen presentation on MHC class II molecules. Here, we show that bone marrow-derived dendritic cells can similarly process and present a foreign antigen expressed by the invasive wild-type and the attenuated PhoP(c) S. typhimurium. This property correlates with equivalent entry and survival efficiencies of both strains in dendritic cells. In addition, Salmonella strains mutated in mgtCB, sseC, and orfL genes required for macrophage survival showed no defect in survival in dendritic cells. Together, these results indicate that uptake of Salmonella by dendritic cells and subsequent antigen processing and presentation do not depend on virulence factors important in macrophages.

Protective immunity induced by Bacillus anthracis toxin-deficient strains.

The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). Six mutant strains that are deficient in the production of one or two of these toxin components have been previously constructed and characterized (C. Pezard, E. Duflot, and M. Mock, J. Gen. Microbiol. 139:2459-2463, 1993). In this work, we examined the antibody response to the in vivo production of PA, LF, and EF in mice immunized with spores of strains producing these proteins. High titers of antibody to PA were observed after immunization with all strains producing PA, while titers of antibodies to EF and LF were weak in animals immunized with strains producing only EF or LF. In contrast, immunization with strains producing either PA and EF or PA and LF resulted in an increased antibody response to EF or LF, respectively. The differing levels of protection from a lethal anthrax challenge afforded to mice immunized with spores of the mutant strains not only confirm the role of PA as the major protective antigen in the humoral response but also indicate a significant contribution of LF and EF to immunoprotection. We observed, however, that PA-deficient strains were also able to provide some protection, thereby suggesting that immune mechanisms other than the humoral response may be involved in immunity to anthrax. Finally, a control strain lacking the toxin-encoding plasmid was unable to provide protection or elicit an antibody response against bacterial antigens, indicating a possible role for pXO1 in the survival of B. anthracis in a host.

Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene.

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. The gene coding for the S-layer protein (sap) was cloned on two contiguous fragments in Escherichia coli, and the complete sequence of the structural gene was determined. The protein, Sap, is composed of 814 residues, including a classical prokaryotic 29-amino-acid signal peptide. The mature form has a calculated molecular mass of 83.7 kDa and a molecular mass of 94 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Sap possesses many charged residues, is weakly acidic, and contains only 0.9% methionine and no cysteine residues. The N-terminal region of Sap shares sequence similarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp alpha protein, and the Bacillus thuringiensis S-layer protein. Electron microscopy observations showed that this S-layer is not observed on B. anthracis cells in which sap has been deleted.

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.

The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence.

Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen). Expression of the toxin genes by B. anthracis is enhanced during growth under elevated levels of CO2. This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a transactivator of anthrax toxin synthesis. Here we show that transcription of atxA does not appear to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new efficient method for gene replacement in B. anthracis, we constructed an atxA-null mutant in which the atxA-coding sequence on pXO1 is replaced with an omega km-2 cassette. Transcription of all three toxin genes is decreased in the absence of atxA. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5' ends mapping to P1 are decreased in the atxA-null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the P1 site is sufficient for atxA-mediated activation of this transcript. The cya and lef genes each have one apparent start site for transcription. Transcripts with 5' ends mapping to these sites are not detected in the atxA-null mutant. The atxA-null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atxA-null mutant-infected mice. These data suggest that the atxA gene product also regulates toxin gene expression during infection.