Functional Metagenomics: A High Throughput Screening Method to Decipher Microbiota-Driven NF-κB Modulation in the Human Gut

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-kappa B signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-kappa 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-kappa 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-kappa 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-alpha and IL-1 beta, were able to activate the reporter system, translating the activation of the NF-kappa B signaling pathway and NF-kappa 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-kappa 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-kappa B regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest

A comparison of random sequence reads versus 16S rDNA sequences for estimating the biodiversity of a metagenomic library

The construction of metagenomic libraries has permitted the study of microorganisms resistant to isolation and the analysis of 16S rDNA sequences has been used for over two decades to examine bacterial biodiversity. Here, we show that the analysis of random sequence reads (RSRs) instead of 16S is a suitable shortcut to estimate the biodiversity of a bacterial community from metagenomic libraries. We generated 10 010 RSRs from a metagenomic library of microorganisms found in human faecal samples. Then searched them using the program BLASTN against a prokaryotic sequence database to assign a taxon to each RSR. The results were compared with those obtained by screening and analysing the clones containing 16S rDNA sequences in the whole library. We found that the biodiversity observed by RSR analysis is consistent with that obtained by 16S rDNA. We also show that RSRs are suitable to compare the biodiversity between different metagenomic libraries. RSRs can thus provide a good estimate of the biodiversity of a metagenomic library and, as an alternative to 16S, this approach is both faster and cheaper

La proline betaiene: transport, biosynthese et accumulation chez Rhizobium meliloti et chez son hote Medicago sativa. Effets d'une contrainte saline

SIGLEINIST T 71503 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Importance of genes I and VI particular CaMV strains in infection and symptom determinants

International audienc

Analyse des determinants genomiques de la severite des symptomes chez le virus de la mosaique du chou-fleur

International audienc

Unique β-Glucuronidase Locus in Gut Microbiomes of Crohn's Disease Patients and Unaffected First-Degree Relatives.

Crohn's disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn's disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn's disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn's disease

Mise en evidence du virus de la mosaique du chou-fleur (CAMV) dans le noyaux des cellules de Brassica pekinensis et de Nicotiana bigelowii

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Gene neighborhood views of H11G1 and C7D2 transporters loci.

<p>We analyzed genes that co-localized with C7D2 or H11G11 transporters from groups 1, 2 and 5 as previously defined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148291#pone.0148291.g002" target="_blank">Fig 2</a>). Gene neighborhood views from microbiomes were obtained using the Gene Mark prediction tool and annotated published genomes. Red box, glucuronide transporter; blue box, BG or GUS β-glucuronidases; black box, AraC family transcriptional regulator; brown box, putative esterase lipase; empty box, putative protein; slashes, sequence end. Homologous sequences are surrounded by same color lines. *: CD patient. Spanish subjects are underlined. This analysis of genes neighboring H11G11 and C7D2 transporters reveals that most CDR have an unexpected C7D2 transporter/GUS β-glucuronidase locus.</p

A conserved NH2-terminal motif in C7D2 transporters from CD related microbiomes.

<p><b>(A)</b> Alignment of NH2-terminal motifs from H11G11 and C7D2 transporters. NH2 terminal motifs were extracted from Expresso (3D Coffee) alignment performed from H11G11 and C7D2 transporters sequences presented <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148291#pone.0148291.g001" target="_blank">Fig 1</a>. * CD unrelated healthy subjects with C7D2 transporter, † no ORF predicted. Black squares indicate C7D2 transporter from CD related microbiomes. Spanish subjects are underlined. <b>(B)</b> Phylogenetic tree based on an alignment restricted to the “FGDFGND” NH2-terminal motif. Neighbor-Joining method was used to perform the tree. Sequences from F1S and C7D2 are absent because the first has no predicted ORF and the second has a truncated NH2-terminal sequence. <b>(C)</b> Proposed pattern for a beta-glucuronidase linked transporter specific of CD related microbiomes. The conserved pattern was determined using C7D2 transporter sequences from CD related microbiomes and published genomes (carrying GND-end motif) as found by the pattern discovery tool PRATT (<a href="http://www.expasy.ch/tools/pratt/" target="_blank">http://www.expasy.ch/tools/pratt/</a>).</p

Phylogenetic tree of H11G11 and C7D2 transporters in CDR and CDU microbiomes.

<p>Analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148291#pone.0148291.g001" target="_blank">Fig 1</a>. Twenty nine protein sequences were retained (>30% identity, > 80% coverage) from the best Blast results for each query. The phylogenetic assignments were performed by searching homologs in published genomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148291#pone.0148291.s011" target="_blank">S3 Table</a>). All protein sequences were aligned using Expresso (3D Coffee) and a Neighbor-Joining tree was performed using MEGA 5.5. Groups are proposed for clusters within high identity percentages (>42% within each group). Sequence names are referred to as: query used/ microbiome sequence name (Metahit and ID 28117 projects). Black and white symbols are used to distinguish CD-related subjects (patients and asymptomatic first-degree relatives; CDR) and CD-unrelated healthy subjects, respectively. The queries used (H11G11 and C7D2 transporters) are underlined. Squares and circles correspond to results using respectively H11G11 and C7D2 transporters as query. This <i>in silico</i> exploration of CDR and CDU microbiomes reveals that H11G11 transporter is mostly present in healthy subjects, whereas C7D2 transporter is recovered in CDR subjects, including unaffected relatives. The C7D2 transporter is plasmid-encoded or present on specific Clostridiale chromosomes.</p