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International audience; Les mammites demeurent une maladie d’importance en élevage laitier, tant par ses conséquences sur l’économie de la filière lait que celles sur le bien-être des vaches et l’utilisation d’antibiotiques. Avec Streptococcus uberis et Staphylococcus aureus, Escherichia coli est un des pathogènes majeurs de mammites, plus particulièrement isolé de mammites cliniques caractérisées par une réponse inflammatoire forte et un fort recrutement de neutrophilesdans le lait. Notre objectif était d’une part d’identifier les propriétés bactériennes nécessaires au déroulement de l’infection et, d’autre part, d’étudier si la reconnaissance des bactéries pouvait être modulée après vaccination.Résultats : L’étude de l’inflammation mammaire induite par différentes souches d’E. coli in vivo dans un modèle murin et in vitro sur des cellules épithéliales mammaires (CEM) a mis en évidence des différences notables entre souches, avec en particulier une réponse pro-inflammatoire in vitro atténuée pour une des souches étudiées. Le criblage d’une banque de mutants aléatoires pour une activité pro-inflammatoire accrue nous a permis de montrer que la présence de l’antigène O à la surface des bactéries réduisait la réponse des CEM et rendait la réponse dépendante du co-récepteur CD14. En parallèle, nous avons cherché à étudier si des anticorps générés suite à une vaccination avec une souche d’E. coli sans antigène O (vaccin J5) pouvait, en reconnaissant des motifs conservés entre souches d’E. coli, améliorer la phagocytose d’E. coli par les neutrophiles. Nos résultats ont montré que tel n’était pas le cas et que l’antigène O à la surface d’E. coli empêchait ces anticorps d’accéder à leur cible, remettant en question l’utilité de l’approche vaccinale J5.Conclusion : Ces travaux permettent de mieux appréhender le rôle de l’antigène O d’E. coli dans le déclenchement de la réponse immunitaire innée et dans la protection contre les mécanismes de phagocytose par les neutrophiles.

International audience

With the extensive use of mobile phones several studies have been realized to understand the effects of radiofrequency exposure on brain activity. Results show changes in the alpha band spectral power. To better understand the cortical structures involved in these changes after exposure at 900 MHz, we carried out electroencephalography and magnetoencephalography recordings followed by the anatomical magnetic resonance imaging. Healthy volunteers were selected according to strict inclusion criteria. Preliminary results on MEG sensor space showed a modification of the alpha band right after exposure. Data analyses of MEG recordings on sensor space and source space are still under process and ready results will be presented at the conference.

International audience; Interleukin 17A-producing T helper cells (Th17) are effector memory CD4+ T cells that are crucial to adaptive immunity to extracellular bacteria. The activities of these cells in the bovine species are not yet defined for want of straightforward cultivation and isolation procedures. We have developed a method to cultivate, expand, sort and maintain in culture bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation (antibodies to CD3 and CD28), we expanded positive CD4+ IL-17A+ T (Th17) cells in a cell culture medium without serum supplemented with TGF-β1 and IL-6. We used IL-2 to expand the cells, which were characterized by intracellular labeling for IL-17A and IFN-γ. Then, we isolated populations of CD4+ T cells producing IL-17A, IFN-γ or both by labeling surface IL-17A with either a complex of biotinylated anti-CD45 antibodies-streptavidin-biotinylated anti-IL-17A antibodies or by direct surface labeling with antibodies to IL-17A, followed by flow cytometry cell sorting. The percentages of surface-labeled IL-17A-secreting cells were quite similar to the percentages of intracellular-labeled IL-17A-producing cells of the same cultures, and the two labeling procedures of live cells yielded similar results. The sorted IL-17A+ cells were restimulated and expanded. After expansion, 80% of the isolated Th17 cells were positive for IL-17A intracellular labeling. The sorted IL-17A+ cells can be frozen, stored and expanded again. The sorted Th17 cells secreted much more IL-17A and IL-17F than did CD4+ IL-17- cells. Notably, most of Th17 cells secreted IFN-γ, although in lower amounts than did CD4+ IL-17- cells. Sorted cells were characterized by transcriptomic profiling. Genes coding for Th17 signature cytokines (IL-17A, IL-17F, IL-26) and transcription factors (RORγt, RORa) were overexpressed in Th17 cells. The techniques developed will make it possible to investigate the phenotypic and functional profiles of bovine Th17 cells along with their stability or plasticity.

International audience

International audience

With the extensive use of mobile phones (MP) several studies have been realized to understand the effects of radiofrequency exposure on brain activity. The results show changes in the alpha band spectral power. To better understand the cortical structures involved in these changes after MP exposure (900 MHz), we carried out electroencephalography and magnetoencephalography recording followed by the anatomical magnetic resonance imaging on healthy volunteers. Participants were selected according to inclusion criteria. They were healthy males or females, aged between 18 and 35, non-smokers and with regular sleep habits. Data analyses of 10 subjects are still under process and ready results will be presented at the conference.

Staphylococcus aureus is the major cause of very severe mastitis of dairy goats. The initial objective of our study was to fine-tune an experimental model of infection of the goat mammary gland with two strains of S. aureus and two lines of goats (low and high somatic cell score lines). Following the challenge, the 10 infected goats divided in two clear-cut severity groups, independently of the S. aureus strain and the goat line. Five goats developed very severe mastitis (of which four were gangrenous) characterized by uncontrolled infection (UI group), whereas the other five kept the infection under control (CI group). The outcome of the infection was determined by 18 h post-infection (hpi), as heralded by the bacterial milk concentration at 18 hpi: more than 107/mL in the UI group, about 106/mL in the CI group. Leukocyte recruitment and composition did not differ between the groups, but the phagocytic killing at 18 hpi efficiency did. Contributing factors involved milk concentrations of α-toxin and LukMF' leukotoxin, but not early expression of the genes encoding the pentraxin PTX3, the cytokines IL-1α and IL-1β, and the chemokines IL-8 and CCL5. Concentrations of TNF-α, IFN-γ, IL-17A, and IL-22 rose sharply in the milk of UI goats when infection was out of control. The results indicate that defenses mobilized by the mammary gland at an early stage of infection were essential to prevent staphylococci from reaching critical concentrations. Staphylococcal exotoxin production appeared to be a consequent event inducing the evolution to gangrenous mastitis.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.

Remerciements :Jean-Luc Touzé and Eric Venturi for help in performing ultrasonography; International audience; Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action has not been documented, and immune responses protecting the mammary gland against E. coli are not completely understood. To improve our knowledge of mammary gland immune protection, cows immunized either intramuscularly or intramammarily with the E. coli P4 were submitted to a homologous mastitis challenge. A third group of mock-immunized cows serve as challenge controls. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical signs and reduction in milk secretion were also contained. This occurred with a modification of the cytokine profile, such as an increase in IFN-γ and a reduction in TNF-α concentrations in milk. Concentrations of IL-17A and IL-22 increased in milk at the onset of the inflammatory response and remained high up to the elimination of bacteria, but concentrations did not differ between groups. Accelerated bacteriological cure was not linked to an increase in the initial efficiency of phagocytosis in milk. Results support the idea that antibodies did not play a major role in the improvement, and that cell-mediated immunity is the key to understanding E. coli vaccine-induced protection of the mammary gland.

International audience; Mastitis remains a major infection of dairy cows and an important issue for dairy farmers and the dairy industry, in particular infections due to Escherichia coli strains. So far, properties specific to E. coli causing mastitis remain ill defined. In an attempt to better understand the properties required for E. coli to trigger mastitis, we used a range of in vitro assays to phenotypically characterize four E. coli strains, including the prototypical E. coli mastitis strain P4, possessing different relative abilities to cause mastitis in a mouse model. Our results indicate that a certain level of serum resistance might be required for colonization of the mammary gland. Resistance to neutrophil killing is also likely to contribute to a slower clearance of bacteria and higher chances to colonize the udder. In addition, we show that the four different strains do induce a pro-inflammatory response by mammary epithelial cells but with different intensities. Interestingly, the prototypical mastitis strain P4 actually induces the less intense response while it is responsible for the most severe infections in vivo. Altogether, our results suggest that different strategies can be used by E. coli strains to colonize the mammary gland and cause mastitis.

We identified a genetic context encoding a transcriptional regulator of the Rgg family and a small hydrophobic peptide (SHP) in nearly all streptococci and suggested that it may be involved in a new quorumsensing mechanism, with SHP playing the role of a pheromone. Here, we provide further support for this hypothesis by constructing a phylogenetic tree of the Rgg and Rgg-like proteins from Gram-positive bacteria and by studying the shp/rgg1358 locus of Streptococcus thermophilus LMD-9. We identified the shp1358 gene as a target of Rgg1358, and used it to confirm the existence of the steps of a quorumsensing mechanism including secretion, maturation and reimportation of the pheromone into the cell. We used surface plasmon resonance to demonstrate interaction between the pheromone and the regulatory protein and performed electrophoretic mobility shift assays to assess binding of the transcriptional regulator to the promoter regions of its target genes. The active form of the pheromone was identified by mass spectrometry. Our findings demonstrate that the shp/rgg1358 locus encodes two components of a novel quorum-sensing mechanism involving a transcriptional regulator of the Rgg family and a SHP pheromone that is detected and reimported into the cell by the Ami oligopeptide transporter.

We characterized the insoluble proteome of Lactococcus lactis using 1D electrophoresis-LC-MS/MS and identified 313 proteins with at least two different peptides. The identified proteins include 89 proteins having a predicted signal peptide and 25 predicted to be membrane-located. In addition, 67 proteins had alkaline isoelectric point values. Using spectra and peptide counts, we compared protein abundances in two different conditions: growth in rich medium, and after transit in the mouse digestive tract. We identified the large mechanosensitive channel and a putative cation transporter as membrane markers of bacterial adaptation to the digestive tract.

Proteins located in the cell envelope are essential in interfacing bacteria with their environment but are still poorly characterised by proteomic approaches due to their low accessibility and solubility. Our goal was to characterise the envelope proteome also called insoluble proteome of L. lactis, used as a model. To do that, we coupled 1D gel electrophoresis and LC-MS/MS analysis. Using spectra and peptide counting, we compared protein abundances in two different environments: during the growth in rich medium (RM) and after transit of the mouse digestive tract (DT). We identified 145 proteins, membrane or cell wall located in L. lactis after the growth in M17 medium. Among them, we identified 46 membrane proteins containing up to 12 transmembrane helixes, lipoproteins and cell wall associated proteins including some with LysM domain. The insoluble fraction also contained many proteins predicted to be cytoplasmic. Most of them belonged to large protein complexes which probably co-purified with the bacterial envelopes. Some others are suspected to be associated to the inner part of the membrane. On the basis of spectral and peptide counting followed by an adapted statistical analysis, we compared the insoluble proteomes from L. lactis after growth in RM or after DT. As expected, we found proteins associated with translation, transcription, DNA replication, transport of nutrients, cell wall biosynthesis, and metabolism more abundant in M17 medium. After transit in digestive tract, L. lactis produced more proteins involved in carbon source diversification, response to osmotic downshift and stress and production of energy. The method used coupling 1D gel electrophoresis, LC-MS/MS and spectra and peptide counting appeared very promising for comparative proteomics of whole bacteria proteomes.

National audience