Benign steatohepatitis (Non Alcoholic Fatty Liver Disease / NAFLD) represents a spectrum of liver diseases encompassing simple fatty infiltration (steatosis), fat and inflammation (non-alcoholic steatohepatitis - NASH), and cirrhosis in the absence of excessive alcohol consumption, viral diseases or other identified etiologies. Due to the high prevalence and to the fact that NAFLD is a common condition that has significant adverse health consequences for those who are afflicted, NAFLD is considered a major public health problem throughout the world. Liver disease has been associated with changes in the intestinal microbiota and specific pathways in which microbiota is involved have been demonstrated to play a role in exacerbation of liver disease. Taking advantage of recently available high throughput sequencing technologies, the three partners involved in the project are currently working in close collaboration to develop a clinically validated biomarker platform to establish quantitative description of gut microbiota and correlations with disease phenotypes. In this context, MetaGenomic Species (MGS, sets of genes predicted to belong to the same microorganism, likely bacterial species) that are associated with NAFLD but are significantly less abundant in advanced liver disease (NASH) were identified. A biomarker is under development based on these results. This project aims at better understanding the biology beyond the biomarkers, i.e. identify which biological functions associated to the biomarkers could explain their potential beneficial effect, as described recently for Faecalibacterium prausnitzii. Since most gut bacterial species cannot easily be cultivated, an innovative strategy will be used. Briefly, specific probes targeting MGS of interest will be used to enrich stool fractions. DNA will then be extracted from these enriched fractions. A high throughput functional screening method of metagenomic libraries will be used to characterize the putative protective effect of molecules that could be encoded by genes belonging to these MGS. Mice will also be colonized with microbiota derived from patients and known to contain low or high proportions of “protective” MGS, then challenged with high fat/fructose diet to explore the effect of these selected microbiota on the onset of steatohepatitis. This project will make it possible for Enterome to validate the technological concept on which its overall activity is based. Indeed, if a functional link is demonstrated between the selected MGS species identified using Enterome’s technology and the development and/or maintenance of the disease, this would fully validate the relevance of the quantitative metagenomic analysis to accurately identify robust biomarkers. In addition the project will generate new IP such as new clones and new molecules of interest that will potentially be further developed in future projects. New technical tools and animal models colonized with MGS of interest will also be useful in future research projects aiming for example at isolating bacterial species corresponding to beneficial MGS. This will be an important asset in a very competitive field of research that has generated lot of interest from big pharmaceutical companies these last few years, thus supporting the international development of the company in a close partnership with its academic partners INRA-Micalis and INRA-Metagenopolis.

Organisms across all kingdoms of life have evolved the ability to turn on specific genetic differentiation programs that confer new properties, such as those defining the different stages of development of multicellular eukaryotic organisms. A well-known and widespread prokaryotic differentiation program is competence for genetic transformation, which provides the cells with the ability to capture, internalize and incorporate exogenous DNA into their genome at sites of homology. As such, this horizontal gene transfer process promotes acquisition of new genetic traits and, consequently, drives evolution of these single-celled microorganisms. The DNA-uptake and processing mechanisms of transformation are conserved among bacteria and rely on some few common effectors that work coordinately with several other species-specific components. Competence is a regulated property and the molecular rules governing its development appear to vary considerably among species. Altogether, competence for genetic transformation appears to be tightly integrated into each particular bacterial life-style and represents a major aspect of their physiology. How competence for genetic transformation is coordinated with other events of the cell cycle remains largely unknown. Here, we will address this question in the major human pathogen Streptococcus pneumoniae (the Pneumococcus), one of the best known and most flexible models for the study of competence for transformation. Remarkably, pneumococcal competence, also termed the X-state, develops abruptly and simultaneously in all cells of an exponentially growing culture and is maintained for a short period of about 20 minutes before rapidly decaying. During this transient period, the cell synthesizes most pieces of the transformasome, a large multiprotein machinery that directs transformation via an ordered series of reactions driving the binding, internalization and integration of exogenous DNA into the recipient genome by homologous recombination. We have previously shown that transformasome assembly in growing pneumococcal cells take place in the cytoplasmic membrane at midcell, the division site. Remarkably, cell growth is transiently delayed in competent pneumococcal cultures. Using single-cell imaging analysis, we found that the cell constriction process is also delayed in competent cells. We inferred that this arrest could reflect the establishment of a cell-division checkpoint that couples the transformation process with the cell cycle, or an interference with cell division owing to assembly of the transformasome at the septum. Another explanation would be that competent pneumococci spatially coordinate remodelling of peptidoglycan with assembly of the transformasome to facilitate its protrusion through the cell wall. The general scientific objectives of this project, named EXStasis for “Exploring the X-state cellular Stasis”, are to discover how competence for genetic transformation provokes a growth arrest in pneumococci and how transformasome assembly is targeted and proceeds at the division site of growing cells. EXStasis is organized into 3 tasks with the goals of understanding the mechanisms developed during pneumococcal competence to take control of the cell-cycle and to assemble a large, membrane-bound machinery spanning the septal membrane and the cell wall at midcell without damaging cell integrity or ability to generate daughter cells. We will address these issues at the molecular level in living cells, by using a multidisciplinary approach combining molecular genetics and proteomics, together with state of the art imaging techniques of different levels of resolution Ultimately, studying how S. pneumoniae coordinates transformation with its life cycle will provide new clues as to the role(s) of competence and transformation in the biology of this pathogenic bacterium.

Surfactants are amphiphilic compounds which reduce the surface tension of water. They are used, among other applications, as detergents, emulsifyers, anti-foaming, and dispersing agents. Most of the surfactants currently used are chemically synthesized. However, interest in biosurfactants of microbial origin has significantly increased during the last decade. Biosurfactants are highly diverse and readily biodegradable. Moreover, the reduction of production costs through fermentation processes and the diversity of their potential applications make these molecules highly attractive in socio-economic terms. Among the microorganims which produce biosurfactants, the Bacillus species are the most known. A new area of research is now emerging which focuses on the search for new biosurfactant molecules, especially lipopeptides, which can be used as phytosanitary products or adjuvants to improve both the ecological fitness and the efficacy of existing biopesticides. Among the set of known lipopeptides, kurstakins produced by Bacillus thuringiensis (Bt) is quite interesting because this is one of the sole cationic lipopeptide family. Bt is the most successful biopesticide used worldwide in agriculture, forest management, and mosquito control. This bacterium, which is recognized as a safe (GRAS) microorganism for environment, animals and humans, is easy to cultivate in large amounts with a low production cost and is fully accepted in organic farming. The capacity of the bacteria to multiply and sporulate in the insect cadaver increases the horizontal transmission of Bt on the leaf surface and might be a key factor for bacterial spreading and, in fine, for the success of the biopesticide treatment. We have recently shown that lipopeptides named kurstakins are essential to the saprophytic properties of Bt, permitting the bacteria to survive and to sporulate in the cadaver of an infected host (an insect larva). Moreover, the results show that these lipopeptides are essential for swarming and biofilm formation, two surface-associated traits requiring biosurfactants. These results indicate that kurstakins might be interesting enhancers of colonisation and persistence of Bacillus strains in the rhizosphere or the phyllosphere. However the main technological barriers for the production and extraction at industrial scale of these compounds are the low level of expression of kurstakin genes and and the resulting low production of the lipopeptide. Our project aims at characterizing and overproducing, on a large scale, a Bt lipopeptide, and at confirming its interest to improve the efficacy of the biopesticide formulations. It is divided into four tasks handled by three teams with complementary competences. The selection of a kurstakin variant, with the most efficient biosurfactant activity and the lowest ecotoxicity, will be performed in task 1. A second task aims to construct a non-sporulating and overproducing strain. This construction will be based on various data including very recent results concerning the regulation of kurstakin expression in Bt obtained by Team 1. A task will focus on the properties and the use of kurstakins as an adjuvant for the formulation of biopesticides. Finally, an important task of this project will be the determination of an appropriate bioprocess for industrial production and purification of kurstakin based on an integrated bioprocess for biosurfactant production recently patented by Team 2. An additional task concerning the commercial development of this technology will be achieved by the INRA-Transfert team.

The intestinal microbiota is a highly complex ecosystem. Many of its members are still not well characterized due to our inability to culture them or to the lack of reference models to study them in detail. Members of the family Coriobacteriaceae are some of these dominant gut bacteria that are still understudied, although they carry important metabolic functions (conversion of bile acids, steroids or phytoestrogens). Recent studies in experimental animal models have shown that the prevalence of 16S rRNA gene sequences assigned to the Coriobacteriaceae is dependent on host genotype and correlates positively with hepatic triglyceride levels or with plasma non-HDL cholesterol levels. Moreover, recent data from the MetaHit consortium showed that one core species of the family, Eggerthella lenta, is linked to the occurrence of type-2 diabetes in human subjects, indicating the need to assess the role of Coriobacteriaceae in metabolic disorders. Bile acid metabolism by bacteria is considered as an important factor influencing host lipid metabolism, yet experimental evidence is sparse. Also, we have recently demonstrated in mice that gut bacteria play important roles in the onset and maintenance of fatty liver disease, but our knowledge of underlying molecular mechanisms is still scant. Similarly, the impact of endogenous corticosteroid metabolism by intestinal bacteria is so far unknown. Thus, it appears crucial to assess in detail microbe/host interactions in the context of host lipid metabolism. In this context, we intend to use strains of Coriobacteriaceae to assess the impact of the bacterial conversion of cholesterol-derived compounds on host lipid metabolism and the development of non-alcoholic hepatic steatosis. We will study host responses (gut and liver) after colonization of germ-free mice by four reference type strains of Coriobacteriaceae. Thereby, we will use various experimental diets as tools to modulate the metabolism of cholesterol-derived products. In addition, we will study the functional adaptation of bacteria to dietary intervention using metatranscriptomic and metabolomic techniques, in order to identify bacterial targets of particular relevance. To bring to the test the importance of these functional targets in an ecological context, we will study the impact of dietary intervention on native Coriobacteriaceae isolated by sorting from the gut content of conventional mice. Finally, the relevance of identified molecular targets (genes or metabolites) will be investigated by targeted quantification in feces from patients with non-alcoholic fatty liver disease vs. healthy controls. Hence, by combining the complementary expertise of research teams in Germany (culture-based microbiology, mouse models and bioanalytics) and France (microbe/host interactions, molecular microbial ecology by metatranscriptomic and functional metagenomics), we intend to characterize the patho-physiological and ecological importance of intestinal Coriobacteriaceae.

The gut microbiota is composed of a huge number of diverse bacterial communities (Bacteroidetes and Firmicutes are the dominant phyla) and of abundant fungi. In humans, the coexistence of resident commensals and host cells play a beneficial role in regulating both energy harvesting from nutrients and host defense. Notably, the intestinal microbiota is able to regulate virulence of enteric bacterial pathogens, including Citrobacter rodentium. However, not all commensals are able to maintain quiescent and protective immunity, arguing for the need to decipher the nature and mechanisms of these processes. The nucleotide-binding oligomerization domain containing protein 2 (Nod2) is thought to play a decisive role in maintaining microbial tolerance and host defense at the intestinal barrier through two adaptor proteins, namely Ripk2 and Card9, but its role in inciting innate and adaptive immunity is complex. Noteworthy, Nod2 and Card9 variants have been associated with Crohn’s disease (CD). More recently, functional data by Partner 2 unveiled a key role of Nod2-driven dysbiosis in predisposing to intestinal inflammation (Couturier-Maillard, et al JCI 2013). More importantly, genetic ablation of either Nod2 (preliminary data of Partner 2) or Card9 (preliminary data of Partner 1 in revision for potential publication in Gastroenterology) results in abnormal host defense against Citrobacter rodentium. Based on such current state of the art, the C. rodentium-driven colitis model is of high interest as appropriate response to such enteric bacterial pathogen requires many major biological processes involved in the maintenance of intestinal homeostasis and that are altered in CD. However, the cellular and molecular mechanism whereby Nod2 and/or Card9 may control pathogen virulence by shaping a non-permissive microbiota has not been investigated yet. Through a multidisciplinary approach, the main objective of the current proposal is therefore to decipher the microbiota-dependent and -independent mechanisms by which the pathogenesis of enteric pathogen bacteria is regulated. To achieve our objective, we envisage an integrated “microbiome/immunology” approach by taking advantage of gnotobiotic animals and by using cre-lox and high-throughput sequencing technologies, as well as developing multidimensional statistical models to decipher the dynamic balance established between the commensal microbiota and the host immune system. Notably, we will use transgenic models of dysbiotic microbiota (ie. Nod2 and Card9 deficient mice) and C. rodentium , as an experimental model microorganism. Whereas most of the studies on this topic so far analyzed either the bacterial gut microbiota or the host response, our ambitious strategy is mainly based on the concomitant analysis of gut microbiota (bacterial but also fungal part) and host compartments. Collectively, our project is highly innovative as it will explore the gut-microbiota crosstalk within the gut in a new dimension by integrating the spatio-temporal host response through combination of up to date genetically engineered mice, gnotobiotic mice, transcriptomics, sequencing technologies and systems biology. Deciphering the complex interactions between the gut microbiota and its host will improve our understanding of human diseases pathogenesis and our discoveries of new therapeutic targets. The intestinal microbiota has been indeed pointed out as a major player in an increasing number of diseases including inflammatory bowel diseases, rheumatoid arthritis, multiple sclerosis, type1 diabetes, obesity or non-alcoholic fatty liver disease.