<em>Enterococcus faecalis</em> Infection Causes Inflammation, Intracellular Oxphos-Independent ROS Production, and DNA Damage in Human Gastric Cancer Cells

Background: Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. Methods: To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing. Results: Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Conclusions: Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-kappa B inflammatory response as well as impaired DNA damage response and cell cycle control gene expression

Enterococcus faecalis Infection and Reactive Oxygen Species Down-Regulates the miR-17-92 Cluster in Gastric Adenocarcinoma Cell Culture

Chronic inflammation due to bacterial overgrowth of the stomach predisposes to the development of gastric cancer and is also associated with high levels of reactive oxygen species (ROS). In recent years increasing attention has been drawn to microRNAs (miRNAs) due to their role in the pathogenesis of many human diseases including gastric cancer. Here we studied the impact of infection by the gram-positive bacteria Enterococcus faecalis (E. faecalis) on global miRNA expression as well as the effect of ROS on selected miRNAs. Human gastric adenocarcinoma cell line MKN74 was infected with living E. faecalis for 24 h or for 5 days or with E. faecalis lysate for 5 days. The miRNA expression was examined by microarray analysis using Affymetrix GeneChip miRNA Arrays. To test the effect of ROS, MKN74 cells were treated with 100 mM tert-Butyl hydroperoxide (TBHP). Following 5 days of E. faecalis infection we found 91 differentially expressed miRNAs in response to living bacteria and 2 miRNAs responded to E. faecalis lysate. We verified the down-regulation of the miR-17-92 and miR-106-363 clusters and of other miRNAs involved in the oxidative stress-response by qRT-PCR. We conclude that only infection by living E. faecalis bacteria caused a significant global response in miRNA expression in the MKN74 cell culture. E. faecalis infection as well as ROS stimulation down-regulated the expression of the miR-17-92 cluster. We believe that these changes could reflect a general response of gastric epithelial cells to bacterial infections

Infection stimulated an up-regulation of several transcripts involved in inflammatory and ROS response pathways.

<p>(A) A section of the GSA result. From the portion of gene sets significantly enriched in MKN74 cells infected for 24 hours, a subset of gene sets were manually selected by association with inflammatory response and response to ROS. The numbers in hard brackets indicate effective number of genes in the gene set. The title of each gene set corresponds to the title given on the MSigDB website. Numbers in colored boxes are adjusted p-values (q-values), black indicate statistical significance at 1% Fdr. (B) Characterization by qRT-PCR of transcripts coding for important cytokines and chemokines after <i>E. faecalis</i> infection for 24 hours (MOI50) and 5 days (MOI10). * denotes significantly different from untreated cells p<0.05.</p

<i>E. faecalis</i> infection induced intracellular ROS production.

<p>MKN74 cells infected with <i>E. faecalis</i> for 30 minutes at MOI50. (A) Representative fluorescence microscope image of MKN74 cells stained with ROS detecting probes (green) and superoxide detecting probes (orange) Scale bars  = 50 µm. (B) Quantification of fluorescence intensity using the LSM 510 software. A statistically significant increased intracellular production of ROS (p<0.01) and superoxide (p<0.02) in the infected cells compared to uninfected control cells was observed. * denotes significant difference. (C) Fluorescence image showing MKN74 plasma membrane (red) and <i>E. faecalis</i> (green) after 4 hours of infection. Flanking images show the YZ and XZ plane. (D) Split image of C. No evidence for bacterial invasion of the cells was observed.</p

Proposed model for how chronic infection by pathogenic bacteria affects gastric epithelial cells.

<p>Proposed model for how chronic infection by pathogenic bacteria affects gastric epithelial cells.</p

Intracellular oxygen consumption in MKN74 cells was independent of oxidative phosphorylation after infection.

<p>MKN74 cells were incubated with or without <i>E. faecalis</i> for 4, 8 or 24 hours. Bacteria were removed and oxygen consumption rate was measured in an XF24 Extracellular Flux Analyzer. (A) ATP turnover, (B) respiratory capacity and (C) oxphos-independant oxygen consumption was determined as described in the text. <b>▪</b> =  Control cells, <b>ο</b>  =  <i>E. faecalis</i> infected cells. (n = 3–6, error bars indicate S.D. ** and *** denotes significant difference p<0.01 and p<0.001 respectively).</p

Infection stimulated an up-regulation of several transcripts involved in DNA damage response pathways.

<p>(A) A section of the GSA result after 24 hours of infection. Gene sets associated with DNA damage repair with same setup as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063147#pone-0063147-g004" target="_blank">figure 4</a> A. (B) Characterization of transcripts coding for important genes involved in MMR after <i>E. faecalis</i> infection for 24 hours (MOI50) and 5 days (MOI10). * denotes significantly different from untreated cells p<0.05.</p