A Functional Metagenomic Strategy to decipher Host-Microbiota cross-talk in the Human Intestinal Tract – FunMetaGen

Using a high throughput screening technique called «Functional Metagenomics« we will identify metagenomic clones carrying large fragments of the genomes of human intestinal Microbiome that can modulate key signaling pathways in intestinal epithelial cells. We have already identified twenty and will continue the high-throughput screening for a maximum of 40 candidates. This step uses a already functional robotic platform.
The second step is to sequence the fragments of bacterial genomes identified, and by a random mutagenesis approach to identify genes involved in these effects. In parallel, a transcriptomic study of human cells exposed to these metagenomic clones will allow us to determine the impact of these interactions and to select the eight most interesting clones for a detailed mechanistic study
The third step consist in deciphering the mechanisms of action using various complementary methods (identification of bacterial molecules or metabolites involved, the putative receptors, signaling pathways, the consequences of these interactions on immune cells).
Three candidates with strong potential pathophysiological relevance will be selected for in vivo studies in mouse models and humans.

The high-throughput screening has been completed and 21,120 metagenomic clones were tested on 14 different reporter cell lines for a total of more than 90 000 tests. At present, 21 metagenomic clones were identified and selected for the research project. In addition, 200 clones additional potential candidates are being validated.
Robotics platform for high throughput screening and screening protocols have been optimized.
Of the 21 clones, 12 were selected for the next steps,that is the characterization of bacterial genes involved. After sequencing, more than half of the clones came from Firmicutes (Gram +), many of which belong to the Clostridium cluster IV and XIVa.
After transposition mutagenesis and re-screening, several loci have been identified as essential for the activation of human intestinal epithelial cells. Steps characterization of the molecules involved in the process is underway and the physiological consequences are analyzed. Currently one of these clones shows major effects in animal models and human biopsies.

FunMetaGen will complement our current scientific understanding of the gut microbiota and its interaction with the host. It will contribute to a better understanding of molecular mechanisms used by commensal intestinal bacteria to shape the intestinal immune system homeostasis and thereby controlling local or otherwise exacerbate inflammatory reactions leading to diseases (Crohn's disease, ulcerative colitis , and obesity).
The advances made in understanding the cross-talk commensal bacteria / host, their implications in these diseases, and the discovery of bioactive compounds should lead to new diagnostic and / or therapeutic.

Two publications are in preparation and several patent applications are also being studied.
The preliminary results and the strategy were presented in several (3) international conferences. This work has also been presented at several (12) invited lectures in symposium/congresses.

Our body surfaces are in close contact with trillions of bacteria, among them 90% inhabit the distal parts of the gastrointestinal tract. One of the major limitations for studying commensal bacteria is our inability to cultivate most microbial species. Recent technical advance, namely metagenomic, has enabled rapid progress in characterizing the genomic and genetic diversities of the gut microbiota. This approach has underscored its highly selected composition, dominated in mammals by bacterial species belonging to few phyla (predominance of Bacteroidetes and Firmicutes). The microbial genome (microbiome) contains ~150 times as many genes as the host genome. Hence, the emerging concept is that the microbiota is an integral component/organ of the host physiology. During millions years of co-evolution, the hosts have developed complex immune mechanisms in order to control this close microbial ecosystem. Accumulating evidence highlighted that the microbiota contributes to local development and tuning of gut homeostasis, shaping of host immune system and maintaining the microbial ecosystem.
Our knowledge on the various contributions of the microbiota to health is still in its infancy and the underlying cellular and molecular mechanisms of its interplay with the host intestinal cells remain poorly understood. The intestinal epithelial cells (IEC) are the first line in contact with microbes and have a key role of in the immune regulation which impact on chronic inflammatory disease. The proposed project will focus on the modulation of the inflammatory processes mediated by the IEC/microbiota dialogue.
We were pioneers in developing a functional metagenomic approach to study microbiota host interactions and have demonstrated that this innovative approach is efficient to identify genes potentially involved in host microbiota cross-talk. Using a high throughput screening platform and the reporter gene-based technology, we will identify bio-active clones from metagenomic libraries able to modulate key targets in intestinal epithelial cells. These targets are the immuno-regulatory relevant genes (TSLP, TGF-beta and RALDH1) and transcription factors NF-?B, AP1, and PPAR?. The study of commensal-host relationship requires bi-directional approaches whereby responses of commensals are identified together with the corresponding host cell responses and relevant effectors. By sequencing and transposon mutagenesis approaches, we will characterize the bacterial genes and the nature of the strain involved in IEC responses. The deep characterization of the cellular responses will be deciphered by human cell transcriptomic analysis. Subsequently, using biochemistry and cell biology, we will identify the bacterial bio-modulator components, the putative host receptor and cellular pathways involved in the interaction with the host cells. Moreover, the integrative study of the dialogue among bacteria, epithelial cells and immune cells in a co-culture approach will allow deciphering the impact of the modulatory components on the host responses. Moreover, we aim at demonstrating the in vivo relevance of the identified mechanisms. For this purpose we will use inflammatory and gnotobiotic mouse models to analyse in vivo the patho-physiological relevance of the identified interactions. Finally, the putative implication of the identified genes in human pathologies will be assessed by monitoring the expression of these genes or the gene-expressing bacteria in healthy donors or in patient with immune-related pathologies.
Altogether this innovative project will allow identifying new bioactive molecular signals from the gut microbiota able to influence the host immune system and characterizing at the molecular level the mechanisms underlying their immunomodulatory properties. Thus, FunMetaGen is geared towards knowledge-based biotechnological developments and nutrition strategies with relevance to health.