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International audience; Persistent neutrophilic inflammation associated with chronic pulmonary infection causes progressive lung injury and, eventually, death in individuals with cystic fibrosis (CF), a genetic disease caused by biallelic mutations in the CF transmembrane conductance regulator (CFTR) gene. Therefore, we examined whether roscovitine, a cyclin-dependent kinase inhibitor that (in other conditions) reduces inflammation while promoting host defense, might provide a beneficial effect in the context of CF. Herein, using CFTR-depleted zebrafish larvae as an innovative vertebrate model of CF immunopathophysiology, combined with murine and human approaches, we sought to determine the effects of roscovitine on innate immune responses to tissue injury and pathogens in the CF condition. We show that roscovitine exerts antiinflammatory and proresolution effects in neutrophilic inflammation induced by infection or tail amputation in zebrafish. Roscovitine reduces overactive epithelial reactive oxygen species (ROS)-mediated neutrophil trafficking by reducing DUOX2/NADPH-oxidase activity and accelerates inflammation resolution by inducing neutrophil apoptosis and reverse migration. It is important to note that, although roscovitine efficiently enhances intracellular bacterial killing of Mycobacterium abscessus in human CF macrophages ex vivo, we found that treatment with roscovitine results in worse infection in mouse and zebrafish models. By interfering with DUOX2/ NADPH oxidase-dependent ROS production, roscovitine reduces the number of neutrophils at infection sites and, consequently, compromises granuloma formation and maintenance, favoring extracellular multiplication of M. abscessus and more severe infection. Our findings bring important new understanding of the immune-targeted action of roscovitine and have significant therapeutic implications for safely targeting inflammation in CF. Copyright

Background Mycobacterium abscessus (Mabs), a rapidly growing Mycobacterium species, is considered an MDR organism. Among the standard antimicrobial multi-drug regimens against Mabs, amikacin is considered as one of the most effective. Parenteral amikacin, as a consequence of its inability to penetrate inside the cells, is only active against extracellular mycobacteria. The use of inhaled liposomal amikacin may yield improved intracellular efficacy by targeting Mabs inside the cells, while reducing its systemic toxicity. Objectives To evaluate the colocalization of an amikacin liposomal inhalation suspension (ALIS) with intracellular Mabs, and then to measure its intracellular anti-Mabs activity. Methods We evaluated the colocalization of ALIS with Mabs in eukaryotic cells such as macrophages (THP-1 and J774.2) or pulmonary epithelial cells (BCi-NS1.1 and MucilAir), using a fluorescent ALIS and GFP-expressing Mabs, to test whether ALIS reaches intracellular Mabs. We then evaluated the intracellular anti-Mabs activity of ALIS inside macrophages using cfu and/or luminescence. Results Using confocal microscopy, we demonstrated fluorescent ALIS and GFP-Mabs colocalization in macrophages and epithelial cells. We also showed that ALIS was active against intracellular Mabs at a concentration of 32 to 64 mg/L, at 3 and 5 days post-infection. Finally, ALIS intracellular activity was confirmed when tested against 53 clinical Mabs isolates, showing intracellular growth reduction for nearly 80% of the isolates. Conclusions Our experiments demonstrate the intracellular localization and intracellular contact between Mabs and ALIS, and antibacterial activity against intracellular Mabs, showing promise for its future use for Mabs pulmonary infections.

International audience; Background: Culture conditions sometimes make it difficult to detect non-tuberculous mycobacteria (NTM), particularly Mycobacterium abscessus, an emerging cystic fibrosis (CF) pathogen. The diagnosis of NTM positive cases not detected by classical culture methods might benefit from the development of a serological assay.Methods: As part of a diagnostic accuracy study, a total of 173 sera CF-patients, including 33 patients with M. abscessus positive cultures, and 31 non-CF healthy controls (HC) were evaluated. Four M. abscessus antigens were used separately, comprising two surface extracts (Interphase (INP) and a TLR2 positive extract (TLR2eF)) and two recombinant proteins (rMAB_2545c and rMAB_0555 also known as the phospholipase C (rPLC)).Results: TLR2eF and rPLC were the most efficient antigens to discriminate NTM-culture positive CF-patients from NTM-culture negative CF-patients. The best clinical values were obtained for the detection of M. abscessus-culture positive CF-patients; with sensitivities for the TLR2eF and rPLC of 81.2% (95% CI:65.7-92.3%) and 87.9% (95% CI:71.9-95.6%) respectively, and specificities of 88.9% (95% CI:85.3-94.8%) and 84.8% (95% CI:80.6-91.5%) respectively. When considering as positive all sera, giving a positive response in at least one of the two tests, and, as negative, all sera negative for both tests, we obtained a sensitivity of 93.9% and a specificity of 80.7% for the detection of M. abscessus-culture positive CF-patients.Conclusion: High antibody titers against TLR2eF and rPLC were obtained in M. abscessus-culture positive CF-patients, allowing us to consider these serological markers as potential tools in the detection of CF-patients infected with M. abscessus.

Mycobacterium abscessus lung infections remain difficult to treat. Recent studies have recognized the power of new combinations of antibiotics, such as bedaquiline and imipenem, although in vitro data have questioned this combination. We report that the efficacy of bedaquiline-imipenem combination treatment relies essentially on the activity of bedaquiline in a C3HeB/FeJ mice model of infection with a rough variant of M. abscessus . The addition of imipenem contributed to clearing the infection in the spleen.

Non‐tuberculous mycobacterium (NTM) infections, such as those caused by Mycobacterium abscessus, are increasing globally. Due to their intrinsic drug resistance, M. abscessus pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against M. abscessus and Mycobacterium tuberculosis, the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against M. abscessus. Structure‐activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD‐88 as a new promising active analogue against M. abscessus. Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half‐life of 3.2 hours., Molecular modelling: Molecular representation of the interaction of PIPD1 and M. abscessus MmpL3 predictive model. On one side, PIPD1 is hydrogen‐bound to the carboxylic acid group of D618 through its amino moiety. On the other side, its hydroxyl group is H‐bound to the hydroxyl of Y219 and a potent interaction with the carboxylic acid group of D258 can occur. A stabilization occurred through π‐π interactions between the toluene moiety of PIPD1 and phenyl rings of F262 and F622.

International audience

Twelve new Cyclophostin and Cyclipostins analogues (CyC19-30) were synthesized, thus extending our series to 38 CyCs. Their antibacterial activities were evaluated against four pathogenic mycobacteria (Mycobacterium abscessus, Mycobacterium marinum, Mycobacterium bovis BCG, and Mycobacterium tuberculosis) and two Gram negative bacteria. The CyCs displayed very low toxicity toward host cells and were only active against mycobacteria. Importantly, several CyCs were active against extracellular M. abscessus (CyC17/CyC18β/CyC25/CyC26) or intramacrophage residing mycobacteria (CyC7(α,β)/CyC8(α,β)) with minimal inhibitory concentrations (MIC50) values comparable to or better than those of amikacin or imipenem, respectively. An activity-based protein profiling combined with mass spectrometry allowed identification of the potential target enzymes of CyC17/CyC26, mostly being involved in lipid metabolism and/or in cell wall biosynthesis. Overall, these results strengthen the selective activity of the CyCs against mycobacteria, including the most drug-resistant M. abscessus, through the cumulative inhibition of a large number of Ser- and Cys-enzymes participating in key physiological processes.

Free-living amoebae might represent an evolutionary niche. In order to get more insights into the potential amoebal training ground forMycobacterium abscessus, we characterized its full transcriptome in amoeba (Ac) and macrophages (Mφ), as well as theMycobacterium chelonaeintra-Ac transcriptome for comparison. Up-regulated genes in Ac allowedM. abscessusto resist environmental stress and induce defense mechanisms, as well as showing switch from carbohydrate carbon sources to fatty acid metabolism. Eleven genes implicated in the adaptation to intracellular stress, were mutated, with all but one confirmed to be involved inM. abscessusintra-Mφ survival. Cloning two of these genes inM. chelonaeincreased its intra-Mφ survival. One mutant was particularly attenuated in Mφ that corresponded to the deletion of an Eis N-acetyl transferase protein (MAB_4532c). Taken together,M. abscessustranscriptomes revealed the intracellular lifestyle of the mycobacteria, with Ac largely contributing to the enhancement ofM. abscessusintra-Mφ survival.

Mycobacterium abscessus is a peculiar rapid-growing Mycobacterium (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as Mycobacterium tuberculosis. A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an M. abscessus transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the MAB_0855 and Mycobacterium chelonae mmpL8 loci. A KO mutant of the MAB_0855 gene, designated herein as mmpL8(MAB), had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mmpL8(MAB) mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8(MAB). Systematic lipid analysis revealed that MmpL8(MAB) was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8(MAB) in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

International audience; Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon (Tn) insertion library of an M. abscessus subspecies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB4 that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.

The coevolution of mycobacteria and amoebae seems to have contributed to shaping the virulence of nontuberculous mycobacteria in macrophages. We identified a pool of genes essential for the intracellular survival of Mycobacterium abscessus inside amoebae and macrophages and discovered a hot spot of transposon insertions within the orthologous ESX-4 T7SS locus. We generated a mutant with the deletion of a structural key ESX component, EccB4. We demonstrate rupture of the phagosomal membrane only in the presence of an intact eccB4 gene. These results suggest an unanticipated role of ESX-4 T7SS in governing the intracellular behavior of a mycobacterium. Because M. abscessus lacks ESX-1, it is tempting to speculate that ESX-4 operates as a surrogate for ESX-1 in M. tuberculosis.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555 , encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis . Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus . Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M . abscessus produces the broad-spectrum β-lactamase Bla Mab . We determine whether inhibition of Bla Mab by avibactam improves the activity of imipenem against M. abscessus . The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of Bla Mab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of Bla Mab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding Bla Mab was induced (20-fold) in the infected macrophages. Inhibition of Bla Mab by avibactam improved the efficacy of imipenem against M. abscessus in vitro , in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of Bla Mab , since the production of the β-lactamase is inducible in macrophages.

Mycobacterium abscessus is an emerging pathogenic mycobacterium involved in pulmonary and mucocutaneous infections, presenting a serious threat for patients with cystic fibrosis (CF). The lack of an efficient treatment regimen and the emergence of multidrug resistance in clinical isolates require the development of new therapeutic strategies against this pathogen. Reverse genetics has revealed genes that are present in M. abscessus but absent from saprophytic mycobacteria and that are potentially involved in pathogenicity. Among them, MAB_3593 encodes MgtC, a known virulence factor involved in intramacrophage survival and adaptation to Mg 2+ deprivation in several major bacterial pathogens. Here, we demonstrated a strong induction of M. abscessus MgtC at both the transcriptional and translational levels when bacteria reside inside macrophages or upon Mg 2+ deprivation. Moreover, we showed that M. abscessus MgtC was recognized by sera from M. abscessus -infected CF patients. The intramacrophage growth (J774 or THP1 cells) of a M. abscessus knockout mgtC mutant was, however, not significantly impeded. Importantly, our results indicated that inhibition of MgtC in vivo through immunization with M. abscessus mgtC DNA, formulated with a tetrafunctional amphiphilic block copolymer, exerted a protective effect against an aerosolized M. abscessus challenge in CF (ΔF508 FVB) mice. The formulated DNA immunization was likely associated with the production of specific MgtC antibodies, which may stimulate a protective effect by counteracting MgtC activity during M. abscessus infection. These results emphasize the importance of M. abscessus MgtC in vivo and provide a basis for the development of novel therapeutic tools against pulmonary M. abscessus infections in CF patients.

Mycobacterium abscessus (Mabs) is a rapidly growing Mycobacterium and an emerging pathogen in humans. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence in zebrafish, which involves the formation of massive serpentine cords, abscesses, and rapid larval death. Generating a cord-deficient Mabs mutant would allow us to address the contribution of cording in the physiopathological signs of the R variant. Herein, a deletion mutant of MAB_4780, encoding a dehydratase, distinct from the β-hydroxyacyl-ACP dehydratase HadABC complex, was constructed in the R morphotype. This mutant exhibited an alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype not only in wild-type but also in immunocompromised zebrafish embryos lacking either macrophages or neutrophils. The abolition of granuloma formation in embryos infected with the dehydratase mutant was associated with a failure to replicate in macrophages, presumably due to limited inhibition of the phagolysosomal fusion. Overall, these results indicate that MAB_4780 is required for Mabs to successfully establish acute and lethal infections. Therefore, targeting MAB_4780 may represent an attractive antivirulence strategy to control Mabs infections, refractory to most standard chemotherapeutic interventions. The combination of a dehydratase assay with a high-resolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors.

International audience; A great number of cystic fibrosis (CF) pathogens such as Pseudomonas aeruginosa, the Burkholderia cepacia and the Mycobacterium abscessus complex raised difficult therapeutic problems due to their intrinsic multiresistance to numerous antibiotics. Vaccine strategies represent one of the key weapons against these multi-resistant bacteria in a number of clinical settings like CF. Different strategies are considered in order to develop such vaccines, linked either to priming the host response, or by exploiting genomic data derived from the bacterium. Interestingly, virulence factors synthesized by various pathogens might serve as targets for vaccine development and have been, for example, evaluated in the context of CF.

This study was performed to determine whether electroporation can be used to enhance the efficacy of a DNA vaccine against pseudorabies virus (PrV) in pigs. Immune responses to PrV were measured in pigs following a single intramuscular injection of plasmids encoding PrV glycoprotein B, with or without electroporation. Plasmid injection coupled with electroporation increased production of specific antibodies against PrV and peripheral blood mononuclear cells proliferated in response to stimulation with PrV glycoproteins. These results show that electroporation can improve the performance of a DNA vaccine against PrV in pigs. However, additional work is required to maximise the effectiveness of the vaccination protocol.

Staphuloccocus aureus enterotoxin A (SEA) is a toxin involved in numerous food poisoning cases. To develop a detection test specific for this toxin, different approaches have been envisaged to obtain the highest antibody titer sera against a peptide sequence from this protein. The present work compares the properties of antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. Different immunization protocols were used: BALB/c mice were immunized either with the peptide or the MAP alone in Freund's adjuvant, co-immunized with the keyhole limpet hemocyanin (KLH) protein or, with the peptide, conjugated to this carrier KLH protein. The extent of reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less than the antibodies raised against the monomeric peptide co-immunized with or conjugated to a carrier protein but more that the antibodies raised against the peptide alone. Inversely, co-immunization of the MAP with the KLH was not able to raise the immune response as it was observed with the monomeric peptide. The results suggest that, for the epitope chosen here, MAP constructs were not the most effective option to induce sera with high levels of antibodies that react with the native protein.

In a previous study, we used bacterial flagellin to deliver antigens such as p27 of Mycobacterium tuberculosis to a host immune system and obtained a potent Th1 response compared to those obtained with Freund's adjuvant and DNA immunization. In the current study, using a POMP91B antigen of Chlamydophila abortus, a human and animal pathogen, as a model, we found that this antigen is unable to promote Th1 response. However, this antigen, unlike others, was able to induce a good Th2 response and IL-4 production after immunization by recombinant protein in Freund's adjuvant or in phosphate buffered saline. Our results suggest that immune response is not only dependent on the immunization adjuvant, but also dependent on the nature of antigen used.

We have recently expressed and characterized the product of the gene Rv2108 of Mycobacterium tuberculosis (MT), the p27 protein. Here, we investigated the immune responses against the p27 protein in the context of different pathogen associated molecular patterns (PAMPs). Different immunization protocols were used. BALB/c mice were immunized either with the p27 recombinant protein in Freund's adjuvant or in phosphate saline buffer (PBS), with a pcDNA3 plasmid containing the gene encoding the p27 protein, or with the Escherichia coli bacteria expressing the p27 protein genetically fused into the flagellin. We found that p27 expressed into the flagellin led to the strongest cellular responses, where we obtained the highest production of IFN-gamma and cell proliferation, an indication of specific Th1-like orientation of the immune response. We confirmed the role of flagellin in this response by using different immunization combinations.

Background Vaccine strategies represent one of the fighting answers against multiresistant bacteria in a number of clinical settings like cystic fibrosis (CF). Mycobacterium abscessus , an emerging CF pathogen, raises difficult therapeutic problems due to its intrinsic antibiotic multiresistance. Methods By reverse vaccinology, we identified M . abscessus phospholipase C (MA-PLC) as a potential vaccine target. We deciphered here the protective response generated by vaccination with plasmid DNA encoding the MA-PLC formulated with a tetra functional block copolymer 704, in CF (ΔF508) mice. Protection was tested against aerosolized smooth and rough (hypervirulent) variants of M. abscessus . Results MA-PLC DNA vaccination (days 0, 21, 42) elicited a strong antibody response. A significant protective effect was obtained against aerosolized M. abscessus (S variant) in ΔF508 mice, but not in wild-type FVB littermates; similar results were observed when: (i) challenging mice with the “hypervirulent” R variant, and; (ii) immunizing mice with purified MA-PLC protein. High IgG titers against MA-PLC protein were measured in CF patients with M. abscessus infection; interestingly, significant titers were also detected in CF patients positive for Pseudomonas aeruginosa versus P. aeruginosa -negative controls. Conclusions MA-PLC DNA- and PLC protein-vaccinated mice cleared more rapidly M. abscessus than β-galactosidase DNA- or PBS- vaccinated mice in the context of CF. PLCs could constitute interesting vaccine targets against common PLC-producing CF pathogens like P. aeruginosa.

DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

Mycobacterium abscessus is responsible for a wide spectrum of clinical syndromes and is one of the most intrinsically drug-resistant mycobacterial species. Recent evaluation of the in vivo therapeutic efficacy of the few potentially active antibiotics against M. abscessus was essentially performed using immunocompromised mice. Herein, we assessed the feasibility and sensitivity of fluorescence imaging for monitoring the in vivo activity of drugs against acute M. abscessus infection using zebrafish embryos. A protocol was developed where clarithromycin and imipenem were directly added to water containing fluorescent M. abscessus -infected embryos in a 96-well plate format. The status of the infection with increasing drug concentrations was visualized on a spatiotemporal level. Drug efficacy was assessed quantitatively by measuring the index of protection, the bacterial burden (CFU), and the number of abscesses through fluorescence measurements. Both drugs were active in infected embryos and were capable of significantly increasing embryo survival in a dose-dependent manner. Protection from bacterial killing correlated with restricted mycobacterial growth in the drug-treated larvae and with reduced pathophysiological symptoms, such as the number of abscesses within the brain. In conclusion, we present here a new and efficient method for testing and compare the in vivo activity of two clinically relevant drugs based on a fluorescent reporter strain in zebrafish embryos. This approach could be used for rapid determination of the in vivo drug susceptibility profile of clinical isolates and to assess the preclinical efficacy of new compounds against M. abscessus .

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-β. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-β in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-β mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-β transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-β promoter, revealed that IFN-β mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-β mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.

The largest and most distinctive class of mycobacteria-specific genes encode a group of 167 proteins of repetitive sequence belonging to the pe and ppe families. The uniqueness of the ppe genes is illustrated by the fact that these genes are restricted to mycobacteria (Cole et al., 1998; Voskuil et al., 2004 (b)). The Rv2108 gene belongs to this family and furthermore is highly specific for the Mycobacterium tuberculosis (Mtb) complex group of mycobacterium (containing notably Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, Mycobacterium pinnipedi, Mycobacterium bovis and M. bovis BCG strain). This gene was described by Chevrier et al., (2000) and used as a molecular probe to develop a rapid test for the detection and identification of this group of mycobacteria. Rv2108 is a gene coding for the protein P27-PPE36, member of the PPE protein family of Mycobacterium tuberculosis, a group of protein thought to be of immunological significance despite the fact that the exact role of the PPE proteins stills unknown.

International audience; A direct, label-free immunosensor was designed for the rapiddetection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions using quartz crystal microbalance with dissipation (QCM-D) as transduction method. The sensing layer including the anti-SEA antibody was constructed by chemisorption of a self-assembled monolayer of cysteamine on the gold electrodes placed over the quartz crystal sensor followed by activation of the surface amino groups with the rigid homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDITC) and covalent linking of binding protein (protein A or protein G). Four anti-SEA antibodies (two of which from commercial source) have been selected to set up the most sensitive detection device. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1000 or 2000 ng ml−1 with a detection limit of 20 ng ml−1. The total time for analysis was 15 min. Using a sandwich type assay, the response was ca. twice higher and consequently the lowest measurable concentration dropped down to 7 ng ml−1 for a total assay time of 25 min.

International audience; DNA vaccination has been widely studied to develop new, alternative, efficient and safe vaccines for humans and animals. Many efforts have been made to increase the immunising potential of these vaccines and three veterinary vaccines are now available on the market. Much work is also being dedicated to develop effective DNA vaccines for humans. However, this new vaccination technique raises issues concerning biosafety due to the nature of the vector, i.e. a DNA molecule that contains sequences of prokaryotic origin (e.g. genes for antibiotic resistance). This review describes the development of the new generation of DNA vectors that are partially or completely devoid of elements of prokaryotic origin and outlines the results of studies on the fate of plasmids after their injection in vivo.

LPS activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. This prompted us to revisit the intriguing issue of CXCL10 production, which we previously showed to be inducible in neutrophils stimulated with LPS and IFN-γ but not with either stimulus alone, contrary to other myeloid cells. We now report that in neutrophils the MyD88-independent pathway is not activated by LPS. Indeed, microarray and real-time PCR experiments showed that neither IFNβ nor IFNβ-dependent genes (including CXCL10) are inducible in LPS-treated neutrophils, in contrast to monocytes. Further investigation into the inability of LPS to promote IFNβ expression in neutrophils revealed that the transcription factors regulating the IFNβ enhanceosome, such as IFN-regulatory factor-3 and AP-1, are not activated in LPS-treated neutrophils as revealed by lack of dimerization, nuclear translocation, confocal microscopy, and inducible binding to DNA. Moreover, we show that the upstream TANK-binding kinase-1 is not activated by LPS in neutrophils. A lack of IFNβ/CXCL10 mRNA expression and IFN-regulatory factor 3 activation was also observed in myeloid leukemia HL60 cells differentiated to granulocytes and then stimulated with LPS, indicating that the inability of neutrophils to activate the MyD88-independent pathway represents a feature of their terminal maturation. These results identify a disconnected activation of the two signaling pathways downstream of TLR4 in key cellular components of the inflammatory and immune responses and help us to better understand the primordial role of neutrophils in host defense against nonviral infections.

In this paper, the polygonal active contour method is adapted to the case of synthetic aperture radar (SAR) images of urban areas. The statistical criterion is modified to be able to deal with multiple images in order to improve the segmentation of buildings. A criterion is proposed and is then implemented and tested over real high resolution SAR images containing urban areas. A discussion about the benefits of this approach is done and further work about 3D statistical active contour is introduced.

Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non-functional, chromosomal flagellin gene (fliC ) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non-motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non-motile, non-transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N-terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN-gamma production and the lack of IL-4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.

Neutrophils, historically known for their involvement in acute inflammation, are also targets for infection by many different DNA and RNA viruses. However, the mechanisms by which they recognize and respond to viral components are poorly understood. Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic mimetic of viral dsRNA that is known to interact either with endosomal TLR3 (not expressed by human neutrophils) or with cytoplasmic RNA helicases such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). In this study, we report that intracellularly administered poly(I:C) stimulates human neutrophils to specifically express elevated mRNA levels encoding type I IFNs, immunoregulatory cytokines, and chemokines, such as TNF-α, IL-12p40, CXCL10, CXCL8, CCL4, and CCL20, as well as classical IFN-responsive genes (IRG), including IFIT1 (IFN-induced protein with tetratricopeptide repeats 1)/IFN-stimulated gene (ISG)56, G1P2/ISG15, PKR (dsRNA-dependent protein kinase), and IFN-regulatory factor (IRF)7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the MAPK-, PKR-, NF-κB-, and TANK (TNF receptor-associated NF-κB kinase)-binding kinase (TBK1)/IRF3-signaling transduction pathways, as illustrated by the use of specific pharmacological inhibitors. Consistent with the requirement of the cytoplasmic dsRNA pathway for antiviral signaling, human neutrophils were found to constitutively express significant levels of both MDA5 and RIG-I, but not TLR3. Accordingly, neutrophils isolated from MDA5-deficient mice had a partial impairment in the production of IFN-β and TNF-α upon infection with encephalomyocarditis virus. Taken together, our data demonstrate that neutrophils are able to activate antiviral responses via helicase recognition, thus acting at the frontline of immunity against viruses.