Enterococcus faecalis infection causes inflammation, intracellular oxphos-independent ROS production, and DNA damage in human gastric cancer cells

PLoS One. 2013 Apr 30;8(4):e63147. doi: 10.1371/journal.pone.0063147. Print 2013.

Abstract

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-κB inflammatory response as well as impaired DNA damage response and cell cycle control gene expression.

Transcript profiling: Array Express accession number E-MEXP-3496.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle / genetics
  • Cell Line, Tumor
  • Cell Proliferation
  • Cluster Analysis
  • DNA Damage*
  • DNA Repair
  • Enterococcus faecalis*
  • Gastric Mucosa / metabolism
  • Gastric Mucosa / microbiology
  • Gastric Mucosa / pathology
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic
  • Gram-Positive Bacterial Infections / complications*
  • Humans
  • Inflammation / genetics
  • Inflammation / metabolism
  • Inflammation / microbiology
  • Intracellular Space / metabolism
  • Mitochondria / genetics
  • Mitochondria / metabolism
  • Models, Biological
  • NF-kappa B / metabolism
  • Oxidative Phosphorylation
  • Reactive Oxygen Species / metabolism*
  • Signal Transduction
  • Stomach Neoplasms / complications
  • Stomach Neoplasms / genetics*
  • Stomach Neoplasms / metabolism*
  • Superoxides / metabolism

Substances

  • NF-kappa B
  • Reactive Oxygen Species
  • Superoxides

Grants and funding

AMDM is supported by a fellowship from Portuguese Science, Technology Foundation. LFH is supported by the Danish MRC. JABS is supported by the Danish Cancer Society, and University of Copenhagen SUND. TMB and OW are supported by a grant from the Novo Nordisk Foundation to the Bioinformatics Centre. CD and LJR are supported by NORDEA-fonden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.