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International audience; Eukaryotic cells are exposed to environmental and endogenous factors that induce DNA damage, thus affecting genomic integrity. The host cells counteract the consequences of lesions by DNA damage response and checkpoint systems that repair DNA structure or trigger cell death when DNA is irreparably damaged. S. aureus, a highly versatile gram-positive bacterium, can cause a multiplicity of human diseases ranging from mild superficial skin to life-threatening disseminated infections. S. aureus is one of the most prevalent pathogen that cause chronic ruminant mastitis that is very difficult to treat. Epithelial cells are able to sense microbes, creating an early line of defense against pathogens. Chronic S. aureus infection is likely to be associated with the internalization of the pathogen by host cells, where bacteria are protected from host defenses. Host cell cycle alteration is one of the highly sophisticated mechanisms pathogens use to hijack the main (defense) functions of the host cells, thus promoting their invasion and colonization. Recently we have shown S. aureus-induced cell cycle alteration in human and bovine epithelial cells.We aimed to investigate whether S. aureus can compromise host genomic integrity.We found that S. aureus can compromise host genomic integrity as indicated by bacteria-induced histone H2AX phosphorylation, a marker of DNA double strand breaks, in human epithelial HeLa and osteoblast-like MG-63 cells. This DNA damage is mediated by alpha phenol-soluble modulins (PSMα1–4), while a specific class of lipoproteins (Lpls), encoded on a pathogenicity island in S. aureus, dampens the H2AX phosphorylation thus counteracting the DNA damage. We demonstrated that this DNA damage is mediated by ROS (reactive oxygen species). DNA damage is followed by the induction of DNA repair that involves the ATM kinase-signaling pathway. An examination of S. aureus strains, isolated from the same patient during acute initial and recurrent bone and joint infections, showed that recurrent strains produce lower amounts of Lpls, induce stronger DNA-damage and prompt the G2/M transition delay to a greater extent that suggest an involvement of these mechanisms in adaptive processes of bacteria during chronicization. Our findings suggest that S. aureus infection has an impact on the genome and epigenome of host cells, which may exert patho-physiological dysfunctions and indicate that the balance between the levels of PSMα and Lpls expression impacts the persistence of the infection.

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions.Importance Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems, and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

The role of Propionibacterium freudenreichii in mitigating inflammation has been a subject of study for many years. Lately, it has been shown that immunomodulation properties are strain- specific and that the major responsible for inflammatory modulation ofimmunomodulation by strain CIRM-BIA129 is the presence of the surface layer protein B (SlpB), which has presented immunomodulation even when expressed in other bacteria. Extracellular vesicles (EVs) are nanosized spherical structures, produced by organisms of all kingdoms, including bacteria. They have been associated with inter-organism communication, pathogenesis, competition, and immunomodulation. Recent studies were aiming to address the role of EVs, in the probiotic effects of bacteria. The properties of P. freudenreichii CIRM-BIA129- derived EVs have been investigated. EVs produced by CIRM-BIA129 cultured in milk ultrafiltrate medium (UF) have been characterized by regarding size and morphology. UF-derived EVs displayed a monodisperse pattern with a modal size of 84.80 ± 2.34 nm. They are composed of a wide variety of proteins, mainly involved in metabolic processes, cellular processes and signaling, and storage and processing of information. A, among these proteins, SlpB was found in high abundance. P. freudenreichii EVs were able to inhibit, in a dose dependent manner, the increase of IL-8 production in HT-29 cells induced with LPS, due to NF-KB pathway inhibition, without causing cell cytotoxicity. EVs produced by a CIRM-BIA129 mutant strain with a knockout for SlpB showed a less efficient reduction in IL-8 production. Results have shown that the environmental conditions are able to modify EVs content and, consequently, their immunomodulatory effects. A change in the growth medium, from UF to YEL (yeast extract-lactate) showed a lower production of EVs, with a slightly larger size. EVs produced in YEL did not perform as well in the inhibition of the NF-KB pathway and had no effect on IL-8 production. Recent results have shown that P. freudenreichii EVs were able to protect Caco-2 cells from inflammation-induced excessive increasing permeability. These Altogether, these results show that Evs produced by beneficial propionibacteria are able to generate immunomodulationtrigger immunomodulation, similar to the parental strain. As for the SlpB role in EVs immunomodulation

International audience; The roles of bacterial extracellular vesicles (EVs) in cell-to-cell signaling are progressively being unraveled. These membranous spheres released by many living cells carry various macromolecules, some of which influence host-pathogen interactions. Bacterial EVs contain RNA, which may serve in communicating with their infected hosts. Staphylococcus aureus, an opportunistic human and animal pathogen, produces EVs whose RNA content is still poorly characterized. Here, we investigated in depth the RNA content of S. aureus EVs. A high-throughput RNA sequencing approach identified RNAs in EVs produced by the clinical S. aureus strain HG003 under different environmental conditions: early- and late-stationary growth phases, and presence or absence of a sublethal vancomycin concentration. On average, sequences corresponding to 78.0% of the annotated transcripts in HG003 genome were identified in HG003 EVs. However, only ~5% of them were highly covered by reads (≥90% coverage) indicating that a large fraction of EV RNAs, notably mRNAs and sRNAs, were fragmented in EVs. According to growth conditions, from 86 to 273 highly covered RNAs were identified into the EVs. They corresponded to 286 unique RNAs, including 220 mRNAs. They coded for numerous virulence-associated factors (hld encoded by the multifunctional sRNA RNAIII, agrBCD, psmβ1, sbi, spa, and isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. Twenty-eight sRNAs were also detected, including bona fide RsaC. The presence of 22 RNAs within HG003 EVs was confirmed by reverse transcription quantitative PCR (RT-qPCR) experiments. Several of these 286 RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the EV RNAs were dramatically affected depending on the growth phase and the presence of vancomycin, whereas much less variations were found in the pool of cellular RNAs of the parent cells. Moreover, the RNA abundance pattern differed between EVs and EV-producing cells according to the growth conditions. Altogether, our findings show that the environment shapes the RNA cargo of the S. aureus EVs. Although the composition of EVs is impacted by the physiological state of the producing cells, our findings suggest a selective packaging of RNAs into EVs, as proposed for EV protein cargo. Our study shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host-pathogen interactions.

International audience; Staphylococcus aureus (S. aureus), a versatile Gram-positive bacterium, is the main cause of bone and joint infections, which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected non-professional phagocytes, human osteoblast-like MG-63 cells, has not yet been fully investigated. To investigate the role of inflammasomes in S. aureus-infected MG-63 cells, the establishement of CASP1–/–MG-63 cell line was carried out using the CRISPR-Cas9 gene editing system. We and others showed that S. aureus can be internalized and survive within professional phagocytes, such as macrophages as well as within non phagocytic epithelial cells or osteoblasts. To study the capacity of intracellular S. aureus to induce inflammasomes formation in MG-63 cells, we confirmed the presence of intracellular bacteria using transmission electron microscopy We show here that S. aureus-infected MG-63 cells but not caspase-1 knock-out CASP1–/–MG-63 cells activate the inflammasome as monitored by the release of mature IL-1β. The effect was strain-dependent. The quorum-sensing system in S. aureus known as the accessory gene regulator (Agr) regulates the expression of many virulence factors including the expression of most S. aureus toxin genes. Of note, the expression of PSMs encoding genes (PSMα 1 to 4, PSMβ 1 and 2, and δ-toxin sometimes called PSMƔ) is tightly controlled by the Agr system. The use of S. aureus LAC (USA300) wild-type strain, its deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated that PSM toxins are involved in inflammasomes-related IL-1β production by infected MG-63 cells. Furthermore, we found that the lack of caspase-1 in CASP1–/–MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1–/– MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance.

International audience; Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines and mitigates the inflammatory response in vitro and in vivo. These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e., modulating NF-κB transcription factor activity and interleukin-8 release), which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications, such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here, we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and, hence, the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical, and functional properties of EVs produced from cells cultivated on laboratory yeast extract lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant and smaller in diameter, and they displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacterium interactions. IMPORTANCE Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in hostmicrobe interactions. Recently, we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

BactInflam IJL focuses on the bacterial components involved in some inflammatory diseases. In particular, in two of them: chronic inflammatory bowel disease (IBD) and mastitis, affecting human health and animal health, respectively.; International audience; Introduction: Bacterial extracellular vesicles (EVs) carry various macromolecules able to affect host-pathogen interactions, such as RNAs. Staphylococcus aureus, an important human and animal pathogen, releases EVs whose RNA content is still unkown. Here, we adress what classes of RNAs compose S. aureus EVs. Methods: S. aureus strain HG003 was cultured in Brain Heart Infusion medium under different in vitro conditions: early- and late-stationary phases, in the presence or absence of a sublethal concentration of vancomycin (0.5 μg/mL). EVswere purified from cell-free culture supernatants using density gradient ultracentrifugation. Bacterial and EV samples were submitted to phenolchloroform RNA extraction, DNAse treatment, and library preparation (Ovation Prokaryotic RNA-Seq, Nugen, rRNA depletion). Sequencing was performed using Illumina, NextSeq500, 75 cycles, single reading, High Output. Results: Particle yields were similar between conditions, however, EVs from late-stationary phases were ∼55% ,larger. On average, 78.0% of HG003 annotated genes were identified in EVs, while only ∼5% presented - 90% read coverage. Highly covered EV RNAs included mRNAs coding for virulence-factors (hld, agrBCD, psmB1, sbi, spa, isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. sRNAs were also detected, including the bona fide rsaC. Interestingly, several of these RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the RNAs in EVs were dramatically affected by growth conditions, whereas much less in the parent cells. Finally, the RNA abundance pattern differed between EVs and parent cells. Summary/Conclusion: To our knowledge, this is the first work characterizing the RNA cargo of S. aureus EVs. Our findings show that EV RNAs are shaped by the environment, and suggest the selective packaging of RNAs into EVs. Finally, this study also shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host-pathogen interactions.

National audience; Les méthodes d’analyse métabarcoding (également appelées métagénomique ciblée ou amplicon) sontde plus en plus utilisées pour étudier la diversité des espèces présentes dans un écosystème (micro organismes,plantes, animaux). Le principe consiste à extraire l’ADN d’un échantillon environnemental puis à amplifier par PCR un fragment cible à l’aide d’un couple d’amorces prédéfini. Ces produits PCR, après ajouts de barcodes(oligonucléotides uniques pour chaque échantillon) et adaptateurs de séquençage, sont ensuite séquencés. Après le séquençage, les séquences sont triées par échantillon grâce aux barcodes puis assignées à des taxons par comparaison avec des séquences de référence. Beaucoup de méthodes et outils d’analyse ont été développés pour obtenir une vision la plus précise possible des écosystèmes étudiés. Les techniques de préparation puis d’analyse des échantillons dépendent de l’écosystème, des questions auxquelles on souhaite répondre et de la technologie de séquençage utilisée. Nous proposons des conseils issus de nos expériences, discussions et lectures bibliographiques afin de guider les lecteurs depuis la planification expérimentale jusqu’à l’analyse des données, en détaillant les points de vigilance à chaque étape.

Propionibacterium freudenreichii (PF) is a food grade bacterium with “generally recognized as safe” status, most commonly used as a starter for the manufacture of fermented dairy products such as Emmental and Leerdammer cheeses. It is also known for its production of vitamin B12 and propionic acid, and is studied for its remarkable anti-inflammatory properties[1,2]. Exopolysaccharide (EPS) production by food bacteria such as PF[3] is receiving increasing attention for their potential applications in improving both texture and the health properties of foodstuff. In this work, we explored the genetic and phenotypic bases for the surface properties of the EPS-producing PF strain P52. In this purpose, the whole genomes of the EPS-producing PF P52 strain and of its spontaneous mutant, with an altered EPS-phenotype, were analyzed to identify genetic determinants for EPS biosynthesis. Using lectin-grafted AFM probes, single-molecule force spectroscopy was used to stretch EPS polymers over immobilized individual bacteria[4,5] of both strains. Results evidenced that spontaneous mutation could lead to 10-fold shortening of the EPS length. Application of the extended Free Join Chain model indicated up to 30×103 Kuhn segments for the EPS-producing mutant, vs 5×103 for the mutant. Figure 1: Typical deflection images and single-molecule force curves obtained by AFM analysis of the wild-type EPS-producing P. freudenreichii strain (left) and of its spontaneous mutant (right) Combination of genetic data and force spectroscopy observations provided insight on the biosynthesis of EPS with variable lengths, therefore on the control of thickness of the bacterial capsule for specific food applications.

International audience

International audience; A complex coacervate is a co-assembled soft material with numerous potential practical applications in food and non-food sectors. It results from the interaction of oppositely charged polyelectrolytes in solution leading to a liquid-liquid phase separation. Examples of complex coacervation between oppositely charged polyelectrolytes is numerous but the complex coacervation involving two oppositely charged proteins (heteroprotein coacervation) was only described recently and exhibits some specificities: it is observed in a narrow range of pH, ionic strength, protein concentration and stoichiometry1. A better knowledge of the mechanism of formation and stabilization of such system is essential to design material with optimized technological properties. In most heteroprotein coacervates studied up to now, the protein molar ratio in the coacervate was constant in the whole domain of formation of the coacervate and it was suggested that it corresponds to the molar ratio of an identified heteroprotein building unit. Surprisingly, in the case of -lactoglobulin( -LG)/lactoferrin(LF) system, the molar ratio in -LG/LF coacervates increased from 4 to almost 8 when the -LG/LF molar ratio in the mixing solution increased suggesting the coexistence of different building units with different -LG/LF molar ratio. By combining protein quantification, fluorescence recovery after photobleaching and solid NMR measurements in the coacervates and docking simulation we suggest that -LG/LF coacervates are formed from 3 different building units with different -LG/LF molar ratio. Change in the proportion of these building units in the coacervates give a plausible explanation of the observed change in -LG/LF molar ratio in the coacervate when changing protein molar ratio in the mixing solution.

Voici la composition du Comité d'Organisation (CO) de JOBIM 2015 Le Comité Logistique (CL) est présidé par : Philippe LEROY (UMR 1095 INRA/UBP, Unité Génétique, Diversité et Ecophysiologie des Céréales - GDEC, Clermont-Ferrand) Eric PEYRETAILLADE (Université d'Auvergne, Unité EA-CIDAM, Clermont-Ferrand) Trois personnes clefs: Secrétariat JOBIM2015 - Manon MARTINET (LB2MN-EA.CIDAM 4678, Université d'Auvergne) Gestion Administrative - Cathy RESSOT (Direction de la Recherche de l’Innovation et de la valorisation, Université d'Auvergne) Gestion Financière - Isabelle DELPIT (Direction de la Recherche de l’Innovation et de la valorisation, Université d'Auvergne) Le Comité Scientifique (CS) est présidé par : Pierre PEYRET (Université d'Auvergne, Unité EA-CIDAM, Clermont-Ferrand) Jérôme SALSE (MR 1095 INRA/UBP, Unité Génétique, Diversité et Ecophysiologie des Céréales - GDEC, Clermont-Ferrand) Événement(s) lié(s) : - 3. Colloque de Génomique Environnementale; Montpellier (FRA) - (2015-10-26 - 2015-10-28)Voici la composition du Comité d'Organisation (CO) de JOBIM 2015Le Comité Logistique (CL) est présidé par : Philippe LEROY (UMR 1095 INRA/UBP, Unité Génétique, Diversité et Ecophysiologie des Céréales - GDEC, Clermont-Ferrand) Eric PEYRETAILLADE (Université d'Auvergne, Unité EA-CIDAM, Clermont-Ferrand)Trois personnes clefs: Secrétariat JOBIM2015 - Manon MARTINET (LB2MN-EA.CIDAM 4678, Université d'Auvergne) Gestion Administrative - Cathy RESSOT (Direction de la Recherche de l’Innovation et de la valorisation, Université d'Auvergne) Gestion Financière - Isabelle DELPIT (Direction de la Recherche de l’Innovation et de la valorisation, Université d'Auvergne) Le Comité Scientifique (CS) est présidé par : Pierre PEYRET (Université d'Auvergne, Unité EA-CIDAM, Clermont-Ferrand) Jérôme SALSE (MR 1095 INRA/UBP, Unité Génétique, Diversité et Ecophysiologie des Céréales - GDEC, Clermont-Ferrand); Microorganisms are considered one of the most important players involved in different environmental processes and services including nutrient cycling, pollutants attenuation as well as plant, animal and human health. In order to understand the functioning of microbial ecosystems and their impact on ecosystem processes we need to accurately assess their composition and response to environmental constraints. The application of high-throughput sequencing technologies to the study of 16S/18S rRNA-genes has revolutionized the characterization of complex microbial ecosystems. However, although it is now possible to generate hundreds of thousands of sequence reads at low costs, the analysis of the obtained data is still challenging: potential source errors including amplification biases, technical contamination, sequencing artifacts and taxonomical affiliation mistakes can lead to misinterpretations of microbial community diversity. Furthermore, progresses in sequencing technologies produce larger number of sequences at lower cost, but many tools are not scalable and pipelines have to be adapted for huge dataset. With the objective of defining best practices to analyze 16S/18S rRNA-gene sequence data, the Metagenomics, species identification, phylogeny pole of INRA was created to put together experience of biologist, bioinformaticians and statisticians of different laboratories.

Mastitis is an inflammatory disease of the mammary gland, which causes huge economic losses in the milk production chain. The involvement of the internal microbiota of the bovine udder in the modulation of pathogens populations and, consequently, of the infection prevalence has never been investigated so far. In this study, the udder microbiota was investigated in 31 quarters (corresponding to 27 cows) whose sanitary status ranged from healthy (i.e. no mastitis during the previous lactations) to susceptible to mastitis (i.e. one to several mastitis episodes during each of the previous lactations). Total bacterial DNA was extracted from foremilk samples and swab samples of the teat canal in each cow. The 16S DNA was PCR-amplified (Variable Regions 3 and 4) and pyrosequenced (GS Flex+, 454 Roche). Phylobiome was analyzed using QIIME, and results were further treated using R, and LEfSe. Clustering of the samples based on their bacterial composition showed a link between udder microbiota and sanitary status. Healthy quarters showed a higher diversity compared to mastitis susceptible ones (Shannon index ~8 and 6 respectively). Discriminant analysis of phylobiomes revealed dysbiosis in mastitis-susceptible quarters and allowed identification of taxonomic markers in relation to mastitis susceptibility. Healthy quarters were associated to a higher proportion of the Clostridia class (including genera such as Ruminococcus, Oscillospira, Roseburia, Dorea…), the Bacteroidetes phylum (Prevotella, Bacteroides, Paludibacter…), and the Bifidobacteriales order (Bifidobacterium) whereas mastitissusceptible quarters showed a higher proportion of the Bacilli class (Staphylococcus) and Chlamydiia class. These results suggest that a diverse and balanced microbiota in the udder contributes to the mammary gland health, likely by exerting a barrier affect with regard to pathogens, as observed in other contexts (e.g. human gut). Mechanisms of interaction between this endogenous microbiota and the mammary gland deserve further investigations, which will help maintain a balanced mammary microbiota e.g. through the development of mammary probiotics.

Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients.Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions.As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype.The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone.

Mastitis is an inflammatory disease of the mammary gland, generally of infectious origin,which causes huge economic losses in the milk production chain and can affect milk yield and quality. The capacity of the internal microbiota of the bovine udder (contained notably in foremilk) to exert a barrier effect with regard to pathogens has never been investigated so far. In this study, we evaluated composition of the bovine udder microbiota in relation to mastitis susceptibility. Udder microbiota was investigated in 31 quarters whose sanitary status ranged from healthy (i.e. no mastitis during the previous lactations) to susceptible to mastitis (i.e. one to several mastitis episodes during each of the previous lactations). Total bacterial DNA was extracted from foremilk sample and swab sample of the teat canal in each quarter. The 16S DNA was PCR-amplified and pyrosequenced. Phylobiome was analyzed using QIIME, and results were further treated using R, and LEfSe. Clustering of the samples based on their bacterial composition showed a link between udder microbiota and sanitary status. Healthy quarters showed a higher diversity compared to mastitis susceptible ones. Discriminant analysis of phylobiomes revealed dysbiosis in mastitis-susceptible quarters and allowed identification of taxonomic markers in relation to mastitis susceptibility. Healthy quarters were associated to a higher proportion of the Clostridia class, the Bacteroidetes phylum and the Bifidobacteriales order whereas mastitis-susceptible quarters showed a higher proportion of the Bacilli class (Staphylococcus) and Chlamydiia class. These results suggest that a diverse and balanced microbiota in the udder contributes to the mammary gland health, likely by exerting a barrier affect with regard to pathogens, as observed in other contexts (e.g.human gut). Mechanisms of interaction between this endogenous microbiota and the mammary gland deserve further investigations, which will help maintain a balanced mammary microbiota e.g. through the development of mammary probiotics.

Dystrophin is involved in Duchenne (DMD) and Becker (BMD) Muscular Dystrophies. A lot of clinical and therapeutic researches are published on DMD but precise molecular role of dystrophin is largely unknown, and consequently the correlation between genotype and phenotype is difficult to establish. However, this relation is essential to offer new therapies to patients. That is why we propose to study function of BMD patient mutated dystrophin to correlate with clinical phenotypes. The database eDystrophin provides an overview of phenotypes associate with these mutations and consequences of each mutation on function and 3D-structures of mutated protein through homology models. The great majority of these mutations are exon deletions located in the central rod domain composed by 24 spectrin-like repeats. The use of eDystrophin, models and molecular dynamics highlights two types of structures at the deletion junction: hybrid repeats that reconstitute a triple coiled-coil like native repeats and fractional repeats that do not reconstitute this structure. Molecular dynamics analysis reveals that fractional repeats may be more deleterious than hybrid repeats. A first correlation between clinical phenotypes and the protein structure is established.!, La dystrophine est une protéine impliquée dans les myopathies de Duchenne (DMD) et de Becker (BMD). Malgré les nombreux travaux de recherche fondamentale et clinique effectués sur ces pathologies, le rôle moléculaire précis de la dystrophine est largement méconnu. A ce jour, la corrélation entre génotype et degré de gravité de ces pathologies est difficile à établir. Cette connaissance s’avère cependant indispensable au développement de nouvelles thérapies. L'objet de cette thèse est l’étude bioinformatique de la fonctionnalité des formes mutées de dystrophine exprimées chez les patients BMD avec l'objectif d'une mise en corrélation avec les phénotypes cliniques. Nous avons dans un premier temps créé la base de données eDystrophin qui apporte des informations sur l’ensemble des mutations BMD en lien avec les phénotypes associés. Des modèles obtenus par homologie ont été construits et une vue d’ensemble des conséquences de chaque mutation sur les propriétés d’interactions et la structure 3D de la protéine est également proposée. La majorité des mutations BMD répertoriées sont des délétions d’exons entiers, ayant pour conséquences des troncatures d’une partie du domaine central de la dystrophine composé de 24 répétitions homologues à la spectrine. Dans la deuxième partie de la thèse, l’utilisation combinée des informations répertoriées dans eDystrophin, et des résultats de modélisation et de dynamique moléculaire a permis de mettre en évidence l’existence de deux catégories de protéines tronquées potentiellement présentes chez les patients BMD : 1) des protéines portant à la jonction de la délétion des répétitions dites « hybrides » qui conservent une structure en faisceau de trois hélices proche des répétitions natives et 2) des protéines portant des répétitions dites « fracturées », de nature hétérogène mais qui ne présentent jamais le motif canonique en triple hélice. L’analyse des dynamiques moléculaires réalisées sur une série d’exemples indique que les mutations qui génèrent des répétitions fracturées sont globalement plus délétères que celles qui génèrent des répétitions hybrides. Une première corrélation a pu être établie avec les phénotypes cliniques d’une cohorte de patients.

International audience; Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD). It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.

Poster