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{"title"=>"Functional metagenomics: A high throughput screening method to decipher microbiota-driven NF-KB modulation in the human gut", "type"=>"journal", "authors"=>[{"first_name"=>"Omar", "last_name"=>"Lakhdari", "scopus_author_id"=>"57191250030"}, {"first_name"=>"Antonietta", "last_name"=>"Cultrone", "scopus_author_id"=>"16041454400"}, {"first_name"=>"Julien", "last_name"=>"Tap", "scopus_author_id"=>"26536756600"}, {"first_name"=>"Karine", "last_name"=>"Gloux", "scopus_author_id"=>"6506579335"}, {"first_name"=>"Françoise", "last_name"=>"Bernard", "scopus_author_id"=>"57198279095"}, {"first_name"=>"S.", "last_name"=>"Dusko Ehrlich", "scopus_author_id"=>"57193642170"}, {"first_name"=>"Fabrice", "last_name"=>"Lefèvre", "scopus_author_id"=>"56539454800"}, {"first_name"=>"Joël", "last_name"=>"Doré", "scopus_author_id"=>"7102317195"}, {"first_name"=>"Hervé M.", "last_name"=>"Blottière", "scopus_author_id"=>"7003533685"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-77958558890", "doi"=>"10.1371/journal.pone.0013092", "sgr"=>"77958558890", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)", "pmid"=>"20927194", "issn"=>"19326203", "pui"=>"359839314"}, "id"=>"f28bf506-277e-385b-ac98-a89f9a421758", "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-kB signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-kB 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-κB 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-κB 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-α and IL-1β, were able to activate the reporter system, translating the activation of the NF-κB signaling pathway and NF-κB 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-κB. Conclusions: We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-κB regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest. © 2010 Lakhdari et al.", "link"=>"http://www.mendeley.com/research/functional-metagenomics-high-throughput-screening-method-decipher-microbiotadriven-nfkb-modulation-h", "reader_count"=>20, "reader_count_by_academic_status"=>{"Researcher"=>3, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>2, "Student > Master"=>3, "Student > Bachelor"=>4}, "reader_count_by_user_role"=>{"Researcher"=>3, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>2, "Student > Master"=>3, "Student > Bachelor"=>4}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Nursing and Health Professions"=>1, "Agricultural and Biological Sciences"=>13, "Medicine and Dentistry"=>1, "Chemistry"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>13}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Australia"=>1}, "group_count"=>1}

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• {"files"=>["https://ndownloader.figshare.com/files/411852", "https://ndownloader.figshare.com/files/411880", "https://ndownloader.figshare.com/files/411911", "https://ndownloader.figshare.com/files/411942", "https://ndownloader.figshare.com/files/411977", "https://ndownloader.figshare.com/files/412015", "https://ndownloader.figshare.com/files/412046", "https://ndownloader.figshare.com/files/412070", "https://ndownloader.figshare.com/files/412092"], "description"=>"<div><h3>Background/Aim</h3><p>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 detection of NF-κB modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota.</p><h3>Methods</h3><p>A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-κB 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-κB modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition.</p><h3>Results</h3><p>The two proinflammatory cytokines, TNF-α and IL-1β, were able to activate the reporter system, translating the activation of the NF-κB signaling pathway and NF-κB 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 <em>Bacteroides</em> strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-κB.</p><h3>Conclusions</h3><p>We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-κB regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest.</p></div>", "links"=>[], "tags"=>["throughput", "decipher", "microbiota-driven", "modulation"], "article_id"=>141360, "categories"=>["Immunology", "Cell Biology", "Microbiology", "Medicine", "Genetics"], "users"=>["Omar Lakhdari", "Antonietta Cultrone", "Julien Tap", "Karine Gloux", "Françoise Bernard", "S. Dusko Ehrlich", "Fabrice Lefèvre", "Joël Doré", "Hervé M. Blottière"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0013092.s001", "https://dx.doi.org/10.1371/journal.pone.0013092.s002", "https://dx.doi.org/10.1371/journal.pone.0013092.s003", "https://dx.doi.org/10.1371/journal.pone.0013092.s004", "https://dx.doi.org/10.1371/journal.pone.0013092.s005", "https://dx.doi.org/10.1371/journal.pone.0013092.s006", "https://dx.doi.org/10.1371/journal.pone.0013092.s007", "https://dx.doi.org/10.1371/journal.pone.0013092.s008", "https://dx.doi.org/10.1371/journal.pone.0013092.s009"], "stats"=>{"downloads"=>34, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Functional_Metagenomics_A_High_Throughput_Screening_Method_to_Decipher_Microbiota_Driven_NF_B_Modulation_in_the_Human_Gut/141360", "title"=>"Functional Metagenomics: A High Throughput Screening Method to Decipher Microbiota-Driven NF-κB Modulation in the Human Gut", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-09-30 00:22:40"}
• {"files"=>["https://ndownloader.figshare.com/files/827243"], "description"=>"<p>a. First screening of 2640 clones of a metagenomic library prepared from the fecal microbiota of CD patients. Lysates were added at 10% vol/vol to HT-29/kb-seap-25. NF-κB activity (expressed as OD_{655 nm}) was normalized to the mean activity of the metagenomic controls (<i>E. coli</i> DH10B + empty fosmid; represented in black). Bacteria growth was expressed as OD_{600 nm}. Standard deviation of the control is represented as horizontal doted line and circle dots represent the 10 active lysates selected for further validation. b. Validation of the effect of 5 activating and 5 inhibiting clones. Metagenomic clones were grown in 8 independent cultures and tested on HT-29/kb-seap-25. Reporter gene activity was assayed after 24 hours stimulation. NF-κB activity (expressed as OD_{655 nm}) was normalized to that of the control (<i>E. coli</i> DH10B + empty fosmid). ** p<0.01 as compared to control.</p>", "links"=>[], "tags"=>["metagenomic", "modulation", "ht-29"], "article_id"=>497598, "categories"=>["Immunology", "Cell Biology", "Microbiology", "Medicine", "Genetics"], "users"=>["Omar Lakhdari", "Antonietta Cultrone", "Julien Tap", "Karine Gloux", "Françoise Bernard", "S. Dusko Ehrlich", "Fabrice Lefèvre", "Joël Doré", "Hervé M. Blottière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013092.g003", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Screening_of_a_metagenomic_library_for_NF_954_B_modulation_using_HT_29_reporter_cells_/497598", "title"=>"Screening of a metagenomic library for NF-κB modulation using HT-29 reporter cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-09-30 02:06:38"}
• {"files"=>["https://ndownloader.figshare.com/files/827098"], "description"=>"<p>Cell surface (a.) and intracellular (b.) expression of different TLRs as assessed by flow cytometry using antibodies to TLR2 to TLR9 (from BD Biosciences) in HT-29/kb-seap-25 cells using a FACScalibur (BD Biosciences). Controls are represented in red. c. Effect of various TLRs ligands on reporter gene activity quantified as OD measured at 655 nm by HT-29/kb-seap-25 cells assessed after 24 hours stimulation. Ligands are for TLR1/2: Pam3CSK4 (1 µg/mL); TLR2: Heat Kiled <i>Listeria monocytogenes</i> (10⁸ cells /mL); TLR3: Poly(I:C) (10 µg/mL); TLR4: <i>E. coli K12</i> LPS (10 µg/mL); TLR5: <i>Salmonella typhimurium</i> Flagellin (10 µg/mL); TLR6/2: FSL1 (1 µg/mL); TLR7: Imiquimod (1 µg/mL); TLR8: ssRNA40 (1 µg/mL); TLR9: ODN2006 (5 µM). All ligands are from Invivogen. *  =  p<0.05.</p>", "links"=>[], "tags"=>["tlrs"], "article_id"=>497459, "categories"=>["Immunology", "Cell Biology", "Microbiology", "Medicine", "Genetics"], "users"=>["Omar Lakhdari", "Antonietta Cultrone", "Julien Tap", "Karine Gloux", "Françoise Bernard", "S. Dusko Ehrlich", "Fabrice Lefèvre", "Joël Doré", "Hervé M. Blottière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013092.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TLRs_expression_and_response_to_TLRs_agonists_/497459", "title"=>"TLRs expression and response to TLRs agonists.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-09-30 02:04:19"}
• {"files"=>["https://ndownloader.figshare.com/files/826912"], "description"=>"<p>a. Response of reporter clones to TNF-α stimulation (10 ng/mL). The signal level (OD_{655 nm}) and the ratio of signal with and without stimulation are displayed. A highly efficient clone HT-29/kb-seap-25 is highlighted (arrow). b. Dose-response of HT-29/kb-seap-25 cells to increasing concentrations of TNF-α or IL-1β. Reporter gene activity was measured after 24 hours stimulation. c. Kinetic of HT-29/kb-seap-25 cells response to incubation with TNF-α (10 ng/mL) for different times. d–f. Inhibition of the TNF-α induction of the NF-κB reporter gene in the HT-29/kb-seap-25 cells. Three inhibitor, BAY 11-7082 (d.), MG132 (e.) and CAPE (f.) were tested in presence (solid line) or absence (doted line) of TNF-α (10 ng/mL). Cells were analyzed for NF-κB activity after 24 hours. In all cases, SEAP activity was measured in the supernatant using Quanti-Blue™ (Invivogen) and was expressed as OD at 655 nm.</p>", "links"=>[], "tags"=>["characterization", "clone"], "article_id"=>497274, "categories"=>["Immunology", "Cell Biology", "Microbiology", "Medicine", "Genetics"], "users"=>["Omar Lakhdari", "Antonietta Cultrone", "Julien Tap", "Karine Gloux", "Françoise Bernard", "S. Dusko Ehrlich", "Fabrice Lefèvre", "Joël Doré", "Hervé M. Blottière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013092.g001", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Selection_and_characterization_of_a_NF_954_B_reporter_clone_of_HT_29_/497274", "title"=>"Selection and characterization of a NF-κB reporter clone of HT-29.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-09-30 02:01:14"}
• {"files"=>["https://ndownloader.figshare.com/files/827399"], "description"=>"<p>a. Effect of 3 selected transposed clones with revertant behavior. Filtered culture supernatants were added at 10% vol/vol to HT-29/kb-seap-25. Control corresponds to <i>E. coli</i> DH10B bearing empty fosmid. *  =  p<0.05. b. Sequence annotation and localization of transposon insertion sites. EZ-TN5 transposon is represented as a grey box. A, B and C corresponds to the insertion region of the transposed clones 52B7/A, 52B7/B and 52B7/C, respectively.</p>", "links"=>[], "tags"=>["bacterial", "genes", "stimulatory"], "article_id"=>497759, "categories"=>["Immunology", "Cell Biology", "Microbiology", "Medicine", "Genetics"], "users"=>["Omar Lakhdari", "Antonietta Cultrone", "Julien Tap", "Karine Gloux", "Françoise Bernard", "S. Dusko Ehrlich", "Fabrice Lefèvre", "Joël Doré", "Hervé M. Blottière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013092.g004", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_the_bacterial_genes_involved_in_the_stimulatory_effect_of_52B7_/497759", "title"=>"Identification of the bacterial genes involved in the stimulatory effect of 52B7.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-09-30 02:09:19"}

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F1000Prime