Dataset Information

Functional Multigenomic Screening of Human-Associated Bacteria for NF-?B-Inducing Bioactive Effectors.

ABSTRACT: The effect of the microbiota on its human host is driven, at least in part, by small-molecule and protein effectors it produces. Here, we report on the use of functional multigenomic screening to identify microbiota-encoded effectors. In this study, genomic DNA from 116 human-associated bacteria was cloned en masse, and the resulting multigenomic library was screened using a nuclear factor-?B reporter (NF-?B) assay. Functional multigenomics builds on the concept of functional metagenomics but takes advantage of increasing advances in cultivating and sequencing human-associated bacteria. Effector genes found to confer NF-?B-inducing activity to Escherichia coli encode proteins in four general categories: cell wall hydrolases, membrane transporters, lipopolysaccharide biosynthetic enzymes, and proteins of unknown function. The compact nature of multigenomic libraries, which results from the ability to normalize input DNA ratios, should simplify screening of libraries using diverse heterologous hosts and reporter assays, increasing the rate of discovery of novel effector genes.IMPORTANCE Human-associated bacteria are thought to encode bioactive small molecules and proteins that play an intimate role in human health and disease. Here, we report on the creation and functional screening of a multigenomic library constructed using genomic DNA from 116 bacteria found at diverse sites across the human body. Individual clones were screened for genes capable of conferring NF-?B-inducing activity to Escherichia coli NF-?B is a useful reporter for a range of cellular processes related to immunity, pathogenesis, and inflammation. Compared to the screening of metagenomic libraries, the ability to normalize input DNA ratios when constructing a multigenomic library should facilitate the more efficient examination of commensal bacteria for diverse bioactivities. Multigenomic screening takes advantage of the growing available resources in culturing and sequencing the human microbiota and generates starting points for more in-depth studies on the mechanisms by which commensal bacteria interact with their human host.

SUBMITTER: Estrela AB

PROVIDER: S-EPMC6867899 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors.

Estrela Andreia B AB Nakashige Toshiki G TG Lemetre Christophe C Woodworth Ian D ID Weisman Jazz L JL Cohen Louis J LJ Brady Sean F SF

mBio 20191119 6

The effect of the microbiota on its human host is driven, at least in part, by small-molecule and protein effectors it produces. Here, we report on the use of functional multigenomic screening to identify microbiota-encoded effectors. In this study, genomic DNA from 116 human-associated bacteria was cloned <i>en masse</i>, and the resulting multigenomic library was screened using a nuclear factor-κB reporter (NF-κB) assay. Functional multigenomics builds on the concept of functional metagenomics ...[more]

PMID: 31744921

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Project description:Dairy products are a sector heavily impacted by food loss, often due to bacterial contaminations. A major source of contamination is associated with the formation of biofilms by bacterial species adopted to proliferate in milk production environment and onto the surfaces of milk processing equipment. Bacterial cells within the biofilm are characterized by increased resistance to unfavorable environmental conditions and antimicrobial agents. Members of the Bacillus genus are the most commonly found spoilage microorganisms in the dairy environment. It appears that physiological behavior of these species is somehow depended on the availability of bivalent cations in the environment. One of the important cations that may affect the bacterial physiology as well as survivability are Zn2+ ions. Thus, the aim of this study was to examine the antimicrobial effect of Zn2+ ions, intending to elucidate the potential of a zinc-based antibacterial treatment suitable for the dairy industry. The antimicrobial effect of different doses of ZnCl2 was assessed microscopically. In addition, expression of biofilm related genes was evaluated using RT-PCR. Analysis of survival rates following heat treatment was conducted in order to exemplify a possible applicative use of Zn2+ ions. Addition of zinc efficiently inhibited biofilm formation by B. subtilis and further disrupted the biofilm bundles. Expression of matrix related genes was found to be notably downregulated. Microscopic evaluation showed that cell elongation was withheld when cells were grown in the presence of zinc. Finally, B. cereus and B. subtilis cells were more susceptible to heat treatment after being exposed to Zn2+ ions. It is believed that an anti-biofilm activity, expressed in downregulation of genes involved in construction of the extracellular matrix, would account for the higher sensitivity of bacteria during heat pasteurization. Consequently, we suggest that Zn2+ ions can be of used as an effective antimicrobial treatment in various applications in the dairy industry, targeting both biofilms and vegetative bacterial cells.

Project description: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.

Dataset Information

Functional Multigenomic Screening of Human-Associated Bacteria for NF-?B-Inducing Bioactive Effectors.

Publications

Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors.

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