Dataset Information

Functional metagenomics: a high throughput screening method to decipher microbiota-driven NF-?B modulation in the human gut.

ABSTRACT: BACKGROUND/AIM: The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-?B signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-?B modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota. METHODS: A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-?B binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn's disease patients was performed to identify NF-?B modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition. RESULTS: The two proinflammatory cytokines, TNF-? and IL-1?, were able to activate the reporter system, translating the activation of the NF-?B signaling pathway and NF-?B inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-?B. CONCLUSIONS: We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-?B regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest.

SUBMITTER: Lakhdari O

PROVIDER: S-EPMC2948039 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Functional metagenomics: a high throughput screening method to decipher microbiota-driven NF-κB modulation in the human gut.

Lakhdari Omar O Cultrone Antonietta A Tap Julien J Gloux Karine K Bernard Françoise F Ehrlich S Dusko SD Lefèvre Fabrice F Doré Joël J Blottière Hervé M HM

PloS one 20100930 9

<h4>Background/aim</h4>The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-κB signaling in IECs. A high throughput cell-based screening assay allowing rapid dete ...[more]

PMID: 20927194

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Project description:Chicken gut microbiota has paramount roles in host performance, health and immunity. Understanding the topological difference in gut microbial community composition is crucial to provide knowledge on the functions of each members of microbiota to the physiological maintenance of the host. The gut microbiota profiling of the chicken was commonly performed previously using culture-dependent and early culture-independent methods which had limited coverage and accuracy. Advances in technology based on next-generation sequencing (NGS), offers unparalleled coverage and depth in determining microbial gut dynamics. Thus, the aim of this study was to investigate the ileal and caecal microbiota development as chicken aged, which is important for future effective gut modulation.Ileal and caecal contents of broiler chicken were extracted from 7, 14, 21 and 42-day old chicken. Genomic DNA was then extracted and amplified based on V3 hyper-variable region of 16S rRNA. Bioinformatics, ecological and statistical analyses such as Principal Coordinate Analysis (PCoA) was performed in mothur software and plotted using PRIMER 6. Additional analyses for predicted metagenomes were performed through PICRUSt and STAMP software package based on Greengenes databases.A distinctive difference in bacterial communities was observed between ilea and caeca as the chicken aged (P < 0.001). The microbial communities in the caeca were more diverse in comparison to the ilea communities. The potentially pathogenic bacteria such as Clostridium were elevated as the chicken aged and the population of beneficial microbe such as Lactobacillus was low at all intervals. On the other hand, based on predicted metagenomes analysed, clear distinction in functions and roles of gut microbiota such as gene pathways related to nutrient absorption (e.g. sugar and amino acid metabolism), and bacterial proliferation and colonization (e.g. bacterial motility proteins, two-component system and bacterial secretion system) were observed between ilea and caeca, respectively (P < 0.05).The caeca microbial communities were more diverse in comparison to ilea. The main functional differences between the two sites were found to be related to nutrient absorption and bacterial colonization. Based on the composition of the microbial community, future gut modulation with beneficial bacteria such as probiotics may benefit the host.

Project description:The intestinal microbiota has many beneficial roles for its host. However, the precise mechanisms developed by the microbiota to influence the host intestinal cell responses are only partially known. The complexity of the ecosystem and our inability to culture most of these micro-organisms have led to the development of molecular approaches such as functional metagenomics, i.e. the heterologous expression of a metagenome in order to identify functions. This elegant strategy coupled to high throughput screening allowed to identify novel enzymes from different ecosystems where culture methods have not yet been adapted to isolate the candidate microorganisms. We have proposed to use this functional metagenomic approach in order to model the microbiota's interaction with the host by combining this heterologous expression with intestinal reporter cell lines. The addition of the cellular component to this functional metagenomic approach introduced a second important source of variability resulting in a novel challenge for high throughput screening. First attempts of high throughput screening with various reporter cell-lines showed a high distribution of the response and consequent difficulties to reproduce the response, impairing an easy and clear identification of confirmed hits. In this study, we developed a robust and reproducible methodology to combine these two biological systems for high throughput application. We optimized experimental setups and completed them by appropriate statistical analysis tools allowing the use this innovative approach in a high throughput manner and on a broad range of reporter assays. We herewith present a methodology allowing a high throughput screening combining two biological systems. Therefore ideal conditions for homogeneity, sensitivity and reproducibility of both metagenomic clones as well as reporter cell lines have been identified and validated. We believe that this innovative method will allow the identification of new bioactive microbial molecules and, subsequently, will promote understanding of host-microbiota interactions.

Dataset Information

Functional metagenomics: a high throughput screening method to decipher microbiota-driven NF-?B modulation in the human gut.

Publications

Functional metagenomics: a high throughput screening method to decipher microbiota-driven NF-κB modulation in the human gut.

Similar Datasets

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