Mathieu Bergé

Metabolic adaptation to a high-fat diet is associated with a change in the gut microbiota

Objective The gut microbiota, which is considered a causal factor in metabolic diseases as shown best in animals, is under the dual influence of the host genome and nutritional environment. This study investigated whether the gut microbiota per se, aside from changes in genetic background and diet, could sign different metabolic phenotypes in mice. Methods The unique animal model of metabolic adaptation was used, whereby C57Bl/6 male mice fed a high-fat carbohydrate-free diet (HFD) became either diabetic (HFD diabetic, HFD-D) or resisted diabetes (HFD diabetes-resistant, HFD-DR). Pyrosequencing of the gut microbiota was carried out to profile the gut microbial community of different metabolic phenotypes. Inflammation, gut permeability, features of white adipose tissue, liver and skeletal muscle were studied. Furthermore, to modify the gut microbiota directly, an additional group of mice was given a gluco-oligosaccharide (GOS)-supplemented HFD (HFD+GOS). Results Despite the mice having the same genetic background and nutritional status, a gut microbial profile specific to each metabolic phenotype was identified. The HFD-D gut microbial profile was associated with increased gut permeability linked to increased endotoxaemia and to a dramatic increase in cell number in the stroma vascular fraction from visceral white adipose tissue. Most of the physiological characteristics of the HFD-fed mice were modulated when gut microbiota was intentionally modified by GOS dietary fibres. Conclusions The gut microbiota is a signature of the metabolic phenotypes independent of differences in host genetic background and diet.

Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater

Biofilms formed in aerobic seawater on stainless steel are known to be efficient catalysts of the electrochemical reduction of oxygen. Based on their genomic analysis, seven bacterial isolates were selected and a cyclic voltammetry (CV) procedure was implemented to check their electrocatalytic activity towards oxygen reduction. All isolates exhibited close catalytic characteristics. Comparison between CVs recorded with glassy carbon and pyrolytic graphite electrodes showed that the catalytic effect was not correlated with the surface area covered by the cells. The low catalytic effect obtained with filtered isolates indicated the involvement of released redox compounds, which was confirmed by CVs performed with adsorbed iron-porphyrin. None of the isolates were able to form electro-active biofilms under constant polarization. The capacity to catalyze oxygen reduction is shown to be a widespread property among bacteria, but the property detected by CV does not necessarily confer the ability to achieve stable oxygen reduction under constant polarization.

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm−2) or 5.34 ± 0.33 log (gu cm−2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ∼300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.

Pneumococcal Competence Coordination Relies on a Cell-Contact Sensing Mechanism

Bacteria have evolved various inducible genetic programs to face many types of stress that challenge their growth and survival. Competence is one such program. It enables genetic transformation, a major horizontal gene transfer process. Competence development in liquid cultures of Streptococcus pneumoniae is synchronized within the whole cell population. This collective behavior is known to depend on an exported signaling Competence Stimulating Peptide (CSP), whose action generates a positive feedback loop. However, it is unclear how this CSP-dependent population switch is coordinated. By monitoring spontaneous competence development in real time during growth of four distinct pneumococcal lineages, we have found that competence shift in the population relies on a self-activated cell fraction that arises via a growth time-dependent mechanism. We demonstrate that CSP remains bound to cells during this event, and conclude that the rate of competence development corresponds to the propagation of competence by contact between activated and quiescent cells. We validated this two-step cell-contact sensing mechanism by measuring competence development during co-cultivation of strains with altered capacity to produce or respond to CSP. Finally, we found that the membrane protein ComD retains the CSP, limiting its free diffusion in the medium. We propose that competence initiator cells originate stochastically in response to stress, to form a distinct subpopulation that then transmits the CSP by cell-cell contact.

Bacterial RadA is a DnaB-type helicase interacting with RecA to promote bidirectional D-loop extension

Homologous recombination (HR) is a central process of genome biology driven by a conserved recombinase, which catalyses the pairing of single-stranded DNA (ssDNA) with double-stranded DNA to generate a D-loop intermediate. Bacterial RadA is a conserved HR effector acting with RecA recombinase to promote ssDNA integration. The mechanism of this RadA-mediated assistance to RecA is unknown. Here, we report functional and structural analyses of RadA from the human pathogen Streptococcus pneumoniae. RadA is found to facilitate RecA-driven ssDNA recombination over long genomic distances during natural transformation. RadA is revealed as a hexameric DnaB-type helicase, which interacts with RecA to promote orientated unwinding of branched DNA molecules mimicking D-loop boundaries. These findings support a model of DNA branch migration in HR, relying on RecA-mediated loading of RadA hexamers on each strand of the recipient dsDNA in the D-loop, from which they migrate divergently to facilitate incorporation of invading ssDNA.

Effects of Ionic Strength on Bacteriophage MS2 Behavior and Their Implications for the Assessment of Virus Retention by Ultrafiltration Membranes

Bacteriophage MS2 is widely used as a surrogate to estimate pathogenic virus elimination by membrane filtration processes used in water treatment. Given that this water technology may be conducted with different types of waters, we focused on investigating the effects of ionic strength on MS2 behavior. For this, MS2 was analyzed suspended in solutions of various ionic strengths, firstly in a batch experiment and secondly during membrane ultrafiltration, and quantified using (i) quantitative RT-PCR (qRT-PCR), which detects the total number of viral genomes, (ii) qRT-PCR without the RNA extraction step, which reflects only particles with a broken capsid (free RNA), and (iii) the Plaque Forming Unit (PFU) method, which detects only infectious viruses. At the beginning of the batch experiments in solutions containing low amounts of salts, a loss of MS2 infectivity (90%) and broken particles (20%) were observed; these proportions did not change during filtration. In contrast, in high ionic strength solutions, bacteriophages kept their biological activity in static conditions but they quickly lost their infectivity during the filtration process. Increasing the ionic strength decreased both the inactivation and the capsid breakup in the feed suspension, and increased the loss of infectivity in filtration retentate, while the amount of MS2 genomes was identical in both experiments. In conclusion, ionic strength effects on MS2 behavior may significantly distort the results of membrane filtration processes and therefore the combination of classical and molecular methods used here is useful for an effective validation of the retention efficiency of ultrafiltration membranes.

Dynamic modeling of Streptococcus pneumoniae competence provides regulatory mechanistic insights

In the human pathogen Streptococcus pneumoniae, the gene regulatory circuit leading to the transient state of competence for natural transformation is based on production of an auto-inducer that activates a positive feedback loop. About one hundred genes are activated in two successive waves linked by a central alternative sigma factor ComX. This mechanism appears to be fundamental to the biological fitness of S. pneumoniae. We have developed a knowledge-based model of the competence cycle that describes average cell behavior. It reveals that the expression rates of the two competence operon, comAB and comCDE, involved in the positive feedback loop must be coordinated to elicit spontaneous competence. Simulations revealed the requirement for an unknown late com gene product that shuts of competence by impairing ComX activity. Further simulations led to the predictions that the membrane protein ComD bound to CSP reacts directly to pH change of the medium and that blindness to CSP during the post-competence phase is controlled by late DprA protein. Both predictions were confirmed experimentally.

O57 Spécificité métagénomique de la flore intestinale, perméabilité intestinale, et métabolisme chez des souris diabétiques. Effets réversibles par les glucooligosaccharides

Introduction Il a ete recemment demontre que les facteurs environnementaux tels que regime riche en graisses et flore intestinale interagissent pour induire une obesite. Cependant, le role de ces facteurs sur le diabete de type 2 (DT2) reste inconnu. Resultats Pour demontrer si le DT2 est associe avec un profil metagenomique independant du regime gras nous avons nourri des souris C57bl6 avec ce regime gras diabetogene (sans obesite) pendant 3 mois et selectionne les souris sensibles (D) et celles resistantes (DR) a l’effet diabetogene du regime. Les profils metagenomiques (DGGE) montrent des differences importantes entre les 2 groupes de souris. Le metagenotype des souris D etait associe a une augmentation de la permeabilite paracellulaire du caecum, une alteration de l’expression des adipokines dans les tissus adipeux, et une augmentation du nombre des lymphocytes dans les depots adipeux. Nous avons ensuite nourri des souris diabetiques par des glucooligosaccharides prebiotiques impactant l’ecologie microbienne. Dans ces conditions, les profils metagenomiques etaient profondement alteres, la permeabilite intestinale etait reduite, la tolerance au glucose et le metabolisme lipidique des adipocytes pratiquement normalises. Conclusion Dans l’ensemble, ces donnees montrent que le phenotype diabetique est associe a une flore intestinale particuliere independamment du statut nutritionnel. L’utilisation de prebiotiques peut controler la flore intestinale et secondairement le diabete.

Dynamic Modeling of Streptococcus pneumoniae Competence Provides Regulatory Mechanistic Insights Into Its Tight Temporal Regulation

In the human pathogen Streptococcus pneumoniae, the gene regulatory circuit leading to the transient state of competence for natural transformation is based on production of an auto-inducer that activates a positive feedback loop. About 100 genes are activated in two successive waves linked by a central alternative sigma factor ComX. This mechanism appears to be fundamental to the biological fitness of S. pneumoniae. We have developed a knowledge-based model of the competence cycle that describes average cell behavior. It reveals that the expression rates of the two competence operons, comAB and comCDE, involved in the positive feedback loop must be coordinated to elicit spontaneous competence. Simulations revealed the requirement for an unknown late com gene product that shuts of competence by impairing ComX activity. Further simulations led to the predictions that the membrane protein ComD bound to CSP reacts directly to pH change of the medium and that blindness to CSP during the post-competence phase is controlled by late DprA protein. Both predictions were confirmed experimentally.

026 La translocation de bactéries intestinales vers les tissus adipeux initie l’inflammation et le diabète induits par un régime gras chez la souris

Rationnel Un regime riche en graisses modifie le microbiote intestinal, initie une inflammation a bas bruit, une resistance a l’insuline et un diabete de type 2. Mais les mecanismes associant microbiote et inflammation metabolique ne sont pas connus. Materiels et methodes Resultats Nos travaux demontrent qu’apres une semaine de regime gras (avant l’apparition du diabete), le nombre de bacteries commensales intestinales viables augmente de plus de 100 fois dans le tissu adipeux et le sang des souris. Cette translocation de bacteries vivantes est prevenue chez des souris dont les recepteurs aux motifs bacteriens Nod1 ou CD14 ont ete invalides. Ces souris ne developpent ensuite pas de diabete. A l’inverse chez la souris ob/ob, la translocation de bacteries viables vers les tissus est spontanement tres augmentee. Cette « bacteriemie metabolique » est notamment associee a une augmentation de l’adherence d’Escherichia coli a la muqueuse intestinale et a sa colocalisation avec les cellules dendritiques de la lamina propria. Afin de diminuer l’adherence et la translocation bacterienne nous avons traite pendant six semaines des souris diabetiques avec le probiotique Bifidobacterium animalis subsp. lactis 420. Ce traitement a reduit l’inflammation metabolique, l’intolerance au glucose et la sensibilite a l’insuline. Conclusion Nos resultats demontrent que le developpement d’un etat diabetique sous regime gras chez la souris, est precede par une augmentation de la translocation bacterienne depuis l’intestin vers les tissus, alimentant ainsi en continu bacteriemie et inflammation metaboliques. De nouvelles perspectives therapeutiques sont ainsi envisagees pour le traitement du diabete de type 2.