Along the food chain, most wet surfaces are covered by biological pellicles called biofilms. Biofilms are composed of spatially organized microorganisms (possibly including pathogens) embedded in an extracellular polymeric matrix. This organic cement can act as a “protective shield” against the action of antimicrobials, thus raising serious problems of pathogens persistence and extra-use of chemical biocides in industrial settings. This is a hot topic for industrialist at a time in which contradictory influences operate: consumers are urgently demanding for safe food (free of bacterial pathogens and toxins), while the European regulation is changing and will likely prohibit some disinfectants in the next few years (Reach, EU directive biocide 98/8/EC). In this context, we recently discovered that planktonic bacilli propelled by flagella are able to tunnel deep within biofilms structures. These bacterial stealth swimmers create transient pores that increase macromolecular transfer within the biofilm. We hypothesized and proved that irrigation of the biofilm by swimmer bacteria can exacerbate killing of biofilm bacteria by facilitating penetration and action of disinfectants from the environment. The objective of this “proof of concept” proposal is to go one step further in the applicability of this new concept by selecting synergetic cocktails of hyper-motile bacteria that can increase biocide efficacy against unwanted biofilms on industrial surfaces. The direct societal and environmental implication of such biological strategy is the drastic reduction of chemical use for the control of pathogens in industrial settings. The long term objective is to entirely replace chemical biocides by natural and specific antimicrobials produced and delivered by bacterial stealth swimmers. The development of this “environmental friendly” approach necessitates new tools to analyze non-invasively such bacterial tunneling dynamics in a biofilm matrix. To address this scientific node, we have assembled in this project a multidisciplinary consortium involving microbiologists, microscopists, computer scientists, together with an industrial partner involved in microbial biotechnology.

To develop effective strategies to prevent childhood obesity it is essential to better understand its causes. Worldwide, the prevalence of overweight and obesity among children has risen dramatically over the last decades. As treatment of established obesity often fails, prevention is highly desirable. Consistent with the developmental origins of health and disease (DOHaD) concept, literature suggests obesity is ‘transmitted’ from mother to child, as the best predictor of the risk of obesity at 8 years of age is the mother's pre-gravid body mass index (BMI). Even though genetic, psychosocial, behavioral, and dietary factors play a role, the contribution of the various channels through which the risk of obesity is transmitted from mother to child remains unclear. In obese humans and obese animal models, intestinal microbiota is altered as compared to lean counterparts. This holds true for obese gestational women. In adults, accumulating evidence suggests that intestinal microbiota is involved in the regulation of energy metabolism and body composition in the host. The transmission of a microbiota, "signature" of the maternal obesity, to her child during early periods of development, could potentially affect his metabolism, since maternal microbiota is the main determinant of colonization of intestine of the child. It was also recently demonstrated in animal models that maternal obesity can induce alterations in the development of neonatal brain structures that regulate appetite and finally, that the microbiota interfere with brain development. We made the original assumption that the transfer of a specific microbiota of obese mother, would be responsible for modulation of hypothalamic nerve circuits and brain stem in the offspring, and contribute to changes in appetite regulation. To explore the specific role of maternal microbiota, we built a scientific strategy combining animal experiments, characterizations of eating behavior and associated neuronal circuits of the offspring, and the performance and computation of high throughout microbiotas analyses with the following specific aims: 1) to demonstrate the impact of maternal obesity-associated microbiota on HT and/or DVC neurodevelopment and eating behavior in offspring; 2) to identify maternal microbiota(s) and bacterial species/gene responsible for altered HT/DVC neurodevelopment and eating behavior in offspring, 3) to prove the causative role of identified bacterial species in alteration of HT/DVC neurodevelopment and eating behavior in offspring. To minimize confounding factors (metabolic status of the obese rat during gestation and lactation, genetic determinism of obese rat strains) we chose to realize, from the first days of life, a vertical transfer of microbiota (intestinal, vaginal or milk microbiota) from dams genetically predisposed to obesity (Sprague Dawley Obese Prone model under high-energy diet) into newborn rats born by caesarean section. The project includes 3 work packages (WP), aimed at: (i) (WP0) managing the project (ii) (WP1) analysing in the progenythe impact of maternal obesity-associated microbiota transfer on brain structure and eating behavior, (iii) (WP2) Identifying the maternal microbiotas involved and bacterial species/genes candidate(s); (iv) (WP3) Assessing the causative role of the identified bacterial specieson neurodevelopmental processes and brain functions related to food intake. This project will 1) produce novel knowledge concerning maternal microbiota as a channel of communication between mother and offspring, and 2) identify early biomarkers (bacterial species or genes) predictive of eating behavior alteration. Such findings could potentially lead to the identification of new targets to develop drugs or nutritional strategies to the mother or newborn for reversing the inheritance of metabolic disorders.

Superoxide dismutases (SOD) are very efficient redox metalloproteins, which protect the cell from oxidative stress. Their catalytic activity of superoxide dismutation can be reproduced by low-molecular weight Mn-complexes, called SOD-mimics (SODm) and the overall characteristics of SODs (tuned redox potential, electrostatic guidance of superoxide, compartmentation in organelles) can serve as a guideline in the design of efficient SODm. Oxidative stress, mainly produced in the mitochondria, is involved in inflammation, including Inflammatory Bowel Diseases (IBD), chosen as the biological target. We wish to develop manganese SODm directly inspired from the mitochondrial Mn-SOD that could exert an anti-inflammatory effect through an intracellular antioxidant activity. These will be studied in cellular models of oxidative stress relevant to IBD. This project will involve several steps. First, using a modular approach, we will conjugate a SODm, already developed by the consortium and known to be active in cells, to various vectors and probes to obtain a series of SOD-mimics with tuned cell-penetration properties or organelle targeting, which could be detected inside cells. We will also develop a new series of peptide-based Mn SODm. We will then determine their intrinsic anti-superoxide activity —kinetics of the reaction with superoxide. Their anti-inflammatory effects on several cell models relevant for IBD, intestinal epithelial cells and monocytes/macrophages, will be evaluated by measuring markers of oxidative stress and inflammation and reactive oxygen species (ROS). We will determine the intracellular content in complexes and their sub-cellular location by innovative imaging techniques. What are the main challenges in this project? This project aims at performing inorganic chemistry inside cells and is thus in line with emergent studies dealing with the control and characterization of small metal complexes in cells. This is a very active new field in inorganic chemical biology for which we need to translate the chemical knowledge we have acquired in the chemist’s round-bottom flasks into cells. Enhancing cell penetration and controlling the targeting of SODm to specific organelles is a real challenge, as is the determination of their speciation (or nature) in cells. Physico-chemical techniques to quantify and map metal cations at the sub-cellular level are now emerging: we will apply conventional fluorescence with tagged complexes but also the most recent techniques, such as X-fluorescence for direct sub-cellular mapping of Mn. Success here will certainly lead to a breakthrough in bio-inorganic chemistry as, at present, little information is available on the subcellular distribution of Mn-complexes SODm. This approach will provide guidelines for the rational improvement of antioxidant SODm with an intracellular activity. The project in inorganic biological chemistry dealing with bio-inspired catalytic SOD-mimics design, evaluation and characterization in cells, and sub-cellular imaging will be developed by a consortium with multidisciplinary expertise.

The 2009 A/H1N1 pandemic has highlighted the unpredictability and potential public health danger posed by influenza viruses. Despite numerous studies, detailed replication mechanism, virus escape mechanism from the immune system, the mechanisms by which species barriers crossing takes place remained unsolved issues. Fundamental answers to these questions are required for improving vaccines, anti-viral drugs and diagnostics and protecting from a new severe pandemic provoked by a pathogenic virus. Influenza A virus (IAV) is a negative-sense single-stranded RNA virus. Its genome consists of eight segments individually encapsidated into ribonucleoprotein (RNP) complexes that are central to the viral life cycle. Each RNP is comprised of a single heterotrimeric RNA polymerase bound to the complementary vRNA termini and multiple copies of the viral nucleoprotein (NP) thus forming an intricate RNA-protein architecture. Crystal structures of isolated fragments of the IAV RNA polymerase subunits as well as NP have been solved, but how the subunits form a biologically functional complex, interact with NP and the viral genome, or modulate interactions with host factors is still unknown. The objective of the collaborative RNAP-IAV project is to understand the molecular mechanisms of human IAV RNP assembly by dissecting the protein-protein and RNA-protein interactions inherent to this intricate architecture. With a complementary, pluridisciplinary and multiscale (cellular-, molecular- and atomic-scale) approach combining cellular, biochemical, structural and computational studies, the 2 partners combine their expertise to address important questions on the structure and function of IAV RNPs, based on solid experimental data both on the nucleoprotein and the polymerase subunits. A new strategy developed by Partner-1 allows the production of the heterotrimeric human IAV RNA polymerase in quantities that are large enough for structural (i.e. crystallography and SAXS) and functional studies, especially to detail the molecular basis of the specific vRNA-polymerase recognition. Recent advances by Partner-1 in generating high quality complexes of partly truncated PA-PB1 heterodimers are especially promising for solving PB1 structure with and without RNA. The skill of Partner-1 in electronic microscopy with a high resolution 5Å resolution will be also extremely valuable and complementary to crystallography and SAXS techniques. Furthermore, the two partners have gained an invaluable skill to control the dynamic oligomeric state of NP that represents another important prerequisite for the achievement of RNAP-IAV project. The structural data obtained by Partner-1 will be exploited by Partner-2 through the design new antivirals candidates targeting protein-protein and RNA-protein interactions based on ongoing work with naproxen, a generic drug targeting NP with novel antiviral properties. Reversed genetics developed by Partner-2 will “translate” the structural results at the cellular scale. In addition, Partner-2 will focus on the nuclear import of neo-synthesized viral PBA-PB1 proteins and their specific interactions with importins nuclear receptors. These cellular studies will take advantage from temperature-sensitive (ts) mutants and revertants on polymerase sub-units, which modulate viral replication and have clear biological significance in terms of adaptation of avian viruses to human hosts. The skills of both partners will be used to combine the different components in order to reconstitute and assemble in vitro a fully functional RNP. This interdisciplinary project will provide new insights into IAV transcription, replication, and how these activities are modified by genetic mutation during host species adaptation and will illuminate fundamental aspects of the viral cycle. The current project also provides new approaches in the field of anti-influenza drug design and attenuated live vaccines.

Chromatin modifications, at the level of either histones or cytosines present in DNA, are fundamental regulators of gene expression in eukaryotes as they control the access of the transcriptional machinery to the targeted promoter regions. When these modifications are transmitted during mitosis, they reprogram the daughter cells without altering the genomic sequence, a process termed « epigenetic ». Recent studies have found that chromatin modifications are induced by bacterial pathogens to interfere with the host transcriptional program. However, the mechanisms at play are poorly characterized. Our project is centered on this new facet of host-pathogen interactions. In line with our published work, we will study the chromatin modifications induced by the intracellular bacterial pathogen Listeria monocytogenes, for which we have identified and gathered preliminary data on several factors targeting chromatin. These factors act by two different strategies: - through activation of specific signalling cascades; - through direct control of chromatin regulators. In this project, we plan to elucidate the molecular basis of these new mechanisms by characterizing bacterial proteins, host factors, chromatin marks and genes reprogrammed. Furthermore, we will determine whether DNA methylation or histone modification profiles imposed by bacterial factors are maintained over time, as chromatin modifications (and parallel gene expression) may be transmitted to daughter cells during cell division. This would imply that an infection leaves an epigenetic mark after pathogen eradication, establishing a memory of infection in parallel to acquired immunity. Besides direct obvious implications of such a discovery on public health, it could shed light on possible mechanisms at play in the etiology of certain unexplained affections, such as autoimmune diseases or certain cancers, via bacterial-mediated epigenetic dysregulation of immune responses. To achieve the goals proposed in this project, we will use cutting-edge technologies, such as chromatin/methyl DNA immunoprecipitation followed by deep sequencing, RNAi screening, tandem affinity purification of chromatin complexes and X-ray crystallography. In addition, this project will greatly benefit from the complementary expertise of the four teams involved (microbiology, cellular biology, genomics, epigenetics/chromatin biochemistry and structural biology), the high quality of the infrastructures, and the numerous tools (stable cell lines, bacterial mutants, purified proteins, knock-out mice), which will guarantee advances in the field. All the elements converge for the success of this project.