Model of host-pathogen Interaction dynamics links In vivo optical imaging and immune responses

Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens

Development of a PbWO4 Detector for Single-Shot Positron Annihilation Lifetime Spectroscopy at the GBAR Experiment

We have developed a PbWO4 (PWO) detector with a large dynamic range to measure the intensity of a positron beam and the absolute density of the ortho-positronium (o-Ps) cloud it creates. A simulation study shows that a setup based on such detectors may be used to determine the angular distribution of the emission and reflection of o-Ps to reduce part of the uncertainties of the measurement. These will allow to improve the precision in the measurement of the cross-section for the (anti)hydrogen formation by (anti)proton-positronium charge exchange and to optimize the yield of antihydrogen ion which is an essential parameter in the GBAR experiment

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

Caractérisation agro-climatique du périmètre sucrier de Ferké 2 au Nord de la Côte d’Ivoire

Objectif : Le présent travail est une contribution aux efforts de recherche appliquée visant à accroître la productivité agricole dans les périmètres sucriers de Ferké par la caractérisation des apports d’eau de pluies au sein de Ferké 2 afin de pouvoir proposer des voies d’amélioration dans la conduite pratique des arrosages.Méthodologie et résultats : Les deux périmètres sucriers de Ferké sont situés au Nord de la Côte d’Ivoire Le climat de la région de Ferké est de type tropical sec avec deux saisons : l’une sèche, de novembre à avril et l’autre humide, de mai à octobre. La caractérisation de la pluviométrie a d’abord consisté à constituer une base de données comportant les 36 cumuls de pluies décadaires correspondant à chacune des 15 années considérées (1999 à 2014). Ensuite, à partir de cette base de données, il s’agissait de déterminer la répartition annuelle des pluies décadaires pour les années sèche, médiane et humide, à l’aide d’une analyse fréquentielle des pluies journalières enregistrées. La variabilité interannuelle de la pluviométrie a été caractérisée par l’indice de Nicholson, une variable centrée-réduite permettant d’atténuer les différences interannuelles afin de faire ressortir l’évolution de la pluviométrie et d’en dégager la tendance climatique pour une série pluviométrique donnéeConclusion et application des résultats : Il est ressorti de cette étude une répartition décadaire de la pluviométrie annuelle très différente selon que l’année est sèche, médiane ou humide avec les fréquences respectivement égales à 8 ans/10, 5ans/10 et 2 ans/10. En outre, les variations de l’indice de Nicholson et de la moyenne mobile des pluies ont montré une atténuation de la pluviométrie sur la période allant de 1999 à 2014. Sur la base de l’évapotranspiration potentielle calculée, le déficit pluviométrique cumulé sur la période sèche concernée par la pratique de l’irrigation était d’environ 200 mm ; celle-ci débute de novembre à mars.Mots clés : climat tropical, analyse fréquentielle, bilan hydrique, évapotranspiration potentielle, front intertropical

Comparative structural and functional analysis of Bunyavirus and Arenavirus cap-snatching Endonucleases

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase

A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories

Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory. The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result. Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing

Theoretical study of "trapping sites" in cryogenic rare gas solids doped with β-dicarbonyl molecules

International audienceA deposition model to simulate the growth of doped rare gas crystals is used. The study involves organic molecules with a single intramolecular hydrogen bond such as malonaldehyde, 2chloromalonaldehyde and acetylacetone as impurities. Different trapping sites were obtained depending on the rare gas properties for a given impurity, and depending on the molecular size and shape for a given crystal. Simulations were carried out by using classical molecular dynamics methods including an anharmonic thermal correction, to take into account the zero point movement of the crystal. The results are correlated to spectroscopic data previously achieved for these systems by steady state IR spectroscopy

The mechanisms used by enteropathogenic Escherichia coli to control filopodia dynamics

Enteropathogenic Escherichia coli (EPEC) subverts actin dynamics in eukaryotic cells by injecting effector proteins via a type III secretion system. First, WxxxE effector Map triggers transient formation of filopodia. Then, following recovery from the filopodial signals, EPEC triggers robust actin polymerization via a signalling complex comprising Tir and the adaptor proteins Nck. In this paper we show that Map triggers filopodia formation by activating Cdc42; expression of dominant-negative Cdc42 or knock-down of Cdc42 by siRNA impaired filopodia formation. In addition, Map binds PDZ1 of NHERF1. We show that Map–NHERF1 interaction is needed for filopodia stabilization in a process involving ezrin and the RhoA/ROCK cascade; expression of dominant-negative ezrin and RhoA or siRNA knock-down of RhoA lead to rapid elimination of filopodia. Moreover, we show that formation of the Tir-Nck signalling complex leads to filopodia withdrawal. Recovery from the filopodial signals requires phosphorylation of a Tir tyrosine (Y474) residue and actin polymerization pathway as both infection of cells with EPEC expressing TirY474S or infection of Nck knockout cells with wild-type EPEC resulted in persistence of filopodia. These results show that EPEC effectors modulate actin dynamics by temporal subverting the Rho GTPases and other actin polymerization pathways for the benefit of the adherent pathogen

Fine-Scale Mapping of the 4q24 Locus Identifies Two Independent Loci Associated with Breast Cancer Risk

Background: A recent association study identified a common variant (rs9790517) at 4q24 to be associated with breast cancer risk. Independent association signals and potential functional variants in this locus have not been explored. Methods: We conducted a fine-mapping analysis in 55,540 breast cancer cases and 51,168 controls from the Breast Cancer Association Consortium. Results: Conditional analyses identified two independent association signals among women of European ancestry, represented by rs9790517 [conditional P = 2.51 × 10−4; OR, 1.04; 95% confidence interval (CI), 1.02–1.07] and rs77928427 (P = 1.86 × 10−4; OR, 1.04; 95% CI, 1.02–1.07). Functional annotation using data from the Encyclopedia of DNA Elements (ENCODE) project revealed two putative functional variants, rs62331150 and rs73838678 in linkage disequilibrium (LD) with rs9790517 (r2 ≥ 0.90) residing in the active promoter or enhancer, respectively, of the nearest gene, TET2. Both variants are located in DNase I hypersensitivity and transcription factor–binding sites. Using data from both The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we showed that rs62331150 was associated with level of expression of TET2 in breast normal and tumor tissue. Conclusion: Our study identified two independent association signals at 4q24 in relation to breast cancer risk and suggested that observed association in this locus may be mediated through the regulation of TET2. Impact: Fine-mapping study with large sample size warranted for identification of independent loci for breast cancer risk