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. 2015 Mar 10;6(2):e00025.
doi: 10.1128/mBio.00025-15.

Bacterial secretions of nonpathogenic Escherichia coli elicit inflammatory pathways: a closer investigation of interkingdom signaling

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Bacterial secretions of nonpathogenic Escherichia coli elicit inflammatory pathways: a closer investigation of interkingdom signaling

Amin Zargar et al. mBio. .

Abstract

There have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenic Escherichia coli strains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq). E. coli BL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.

Importance: The symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to "communicate" with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

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Figures

FIG 1
FIG 1
Interkingdom communication between the microbiome and the host in the GI tract. (a) Quorum-sensing (QS) molecules coordinate actions among bacteria. (b) Secretomes of bacteria, including QS molecules, affect the host's cellular machinery. (c) Epithelial cells secrete signals to neighboring and distant cells through signaling molecules. (d) Soluble factors secreted by the host affect bacteria.
FIG 2
FIG 2
(A) HCT-8 epithelial cells were grown to confluence and then incubated with BL21, W3110, or medium alone in the upper chamber of a transwell. After 6 h of incubation, the RNAs of the epithelial cells were extracted and sequenced. (B) Downstream RNA-Seq pipeline for analysis of sequencing data (brown boxes indicate the open-source program). (C) Results of mapping HCT-8 NGS transcripts to a RefSeq annotated human genome, hg19. Five biological replicates were performed with the software TopHat. (D) Genes differentially expressed as determined by using the software DESeq. There were 542 DE genes between HCT-8 cells incubated with BL21 and those incubated with blank medium, and 481 DE genes between HCT-8 cells incubated with W3110 and those incubated with blank medium. We found 280 DE genes between HCT-8 cells incubated with BL21 compared to those incubated with W3110. Of the 542 DE genes in incubations with BL21 compared to blank medium and 481 DE genes in incubations with W3110 compared to blank medium alone were 214 differentially expressed genes common to incubations with either bacteria compared to blank medium.
FIG 3
FIG 3
(A) Activation of the cytokine-cytokine receptor interaction pathway in incubations with BL21 or W3110. The schematic shows cytokines (ovals) and cytokine receptors (polygons) upregulated only by incubation with BL21 (blue) or only by incubation with W3110 (red). Incubations with either E. coli strain (purple) or with no change in regulation by either E. coli strain (gray) is also shown. (B) Schematic of genes involved in the canonical NF-κB pathway, adapted from the KEGG. Gene expression levels upregulated (green) and unaffected (gray) by incubations with both BL21 and W3110 compared to expression levels in medium alone are shown.
FIG 4
FIG 4
Heatmap of the 25 most upregulated (green) and downregulated (red) genes in HCT-8 in incubations with BL21 compared to their levels of expression in medium alone and in incubations with W3110 compared to blank medium. The trace line (white) indicates the direction and extent of differential expression. Differential expression levels are similar between incubations of BL21 and W3110, and 100% of differential expression is regulated in the same manner (i.e., up- or downregulated). DESeq was used to identify differential expression, and all genes listed have a Benjamini-Hochberg-adjusted P of <0.05. Cytokines, including IL-8 (red text), were among the genes most upregulated.
FIG 5
FIG 5
NGS sequenced reads mapped the annotated IL-8 gene as visualized in the Integrative Genome Viewer (IGV). The IL-8 gene is shown at the bottom, with four exons separated by three introns. Each read is represented by a blue square, and the abundance of reads under each condition (BL21, W3110, or medium alone) is shown. HCT-8 incubations with W3110 show a greater abundance of IL-8 transcription than incubations with medium alone, while incubations with BL21 illustrate higher levels than W3110. One representative replicate sample of each condition is shown.
FIG 6
FIG 6
HCT-8 cells were incubated with AI-2 at 50, 150, and 400 μM for 6, 12, and 24 h; expression levels are normalized to those with medium alone. At early times (6 and 12 h), incubations with AI-2 result in an upregulation of IL-8 gene expression compared to expression in medium alone, while at 24 h, IL-8 expression levels are downregulated compared to levels in medium alone. qPCR fold level changes are shown. †, P < 0.10; *, P < 0.05; **, P < 0.01.

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