Figure 1.
E. faecalis infection induced intracellular ROS production.
MKN74 cells infected with E. faecalis 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 E. faecalis (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.
Figure 2.
Intracellular oxygen consumption in MKN74 cells was independent of oxidative phosphorylation after infection.
MKN74 cells were incubated with or without E. faecalis 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. ▪ = Control cells, ο = E. faecalis infected cells. (n = 3–6, error bars indicate S.D. ** and *** denotes significant difference p<0.01 and p<0.001 respectively).
Figure 3.
E. faecalis infection increased mitochondrial mutations in MKN74 cells.
Mutations detected in the mtDNA D-loop region after infection. MKN74 cells were infected with E. faecalis at MOI 10 for 5 days. Infected MKN74 cell cultures had a significantly higher number of total mutations and transition mutations than non-infected control cell cultures. * denotes significantly different from untreated cells p<0.05.
Figure 4.
Infection stimulated an up-regulation of several transcripts involved in inflammatory and ROS response pathways.
(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 E. faecalis infection for 24 hours (MOI50) and 5 days (MOI10). * denotes significantly different from untreated cells p<0.05.
Figure 5.
Infection stimulated an up-regulation of several transcripts involved in DNA damage response pathways.
(A) A section of the GSA result after 24 hours of infection. Gene sets associated with DNA damage repair with same setup as figure 4 A. (B) Characterization of transcripts coding for important genes involved in MMR after E. faecalis infection for 24 hours (MOI50) and 5 days (MOI10). * denotes significantly different from untreated cells p<0.05.
Table 1.
Genes involved in the inflammatory response of MKN74 cells identified by microarray analysis.
Figure 6.
Infection caused a down-regulation of transcripts involved in cell cycle control pathways, and E. faecalis lysate slowed down cell proliferation.
(A) A section of the GSA result after 24 hours of infection. Gene sets associated with cell cycle control with same setup as figure 4 A. (B) MKN74 growth assay. The fold change in number of cells relative to day zero is shown. The growth of MKN74 cells, infected with viable E. faecalis at a MOI of 10 or with different concentrations of bacterial lysate, was followed for a period of five days. Uninfected cells served as controls. Cell density was measured by crystal violet absorbance. ** and *** denotes significant difference p<0.01 and p<0.001 respectively.
Figure 7.
Proposed model for how chronic infection by pathogenic bacteria affects gastric epithelial cells.