Metabolic response of Enterococcus faecalis to oxidative stress

Abstract

Enterococcus faecalis is a lactic acid bacterium once known only as a prominent constituent of the human gut microflora and a popular agent used in industrial fermentation processes. However during the last few years, it has emerged as an important opportunistic pathogen, particularly in the nosocomial context, due to its proficient acquisition of resistance to a large repertoire of antibiotics. Understanding how this bacterium responds to oxidative stress is paramount in gaining insights into its pathology; as tolerance to oxidative stress plays a vital role in the survival of E.faecalis under oxygen-rich environment and in the face of immune insults and killing by bactericidal antibiotics. Therefore the aim of this study was to explore the metabolic changes occurring in E. faecalis when exposed to oxidative stress in the form of hydrogen peroxide (H₂O₂), with specific focus on how this response could be influenced by aeration and growth rate. Utilising tools from the rising field of metabolomics and fluxomics, profiles of intracellular and extracellular metabolites were obtained using gas chromatography-mass spectrometry (GC-MS) and metabolic flux analysis was carried out using 13C-labelled glucose. Differential metabolite profiles combined with 13C-labelling analysis emphasised that response of E. faecalis to H₂OO₂ is highly-dependent on the cell's growth rate and the surrounding environment. Cells with low growth rate or under aerobic growth appeared to have already activated some degree of stress response, thus allowing them to readily adapt to the subsequent change in redox state. The redistribution of carbohydrate flux was demonstrated to have occurred as part of metabolic response to H₂O₂ as the cell enhances NADPH regeneration by up-regulating pentose phosphate pathway. This ties in closely with the greater demand for glutathione and methionine, which both exert anti-oxidative activities with associated NADPH-dependent enzymes. Furthermore, we have highlighted the up-regulation of branched-chain amino acid metabolism inn E. faecalis in the presence of H₂O₂ and the cell's particular reliance on exogenous sources to obtain these amino acids. We have also made the novel discovery of two metabolites, 2-aminobutyric acid and 2-hydroxybutyric acid in E. faecal is that may play an important part during oxidative stress response. Finally, we have re-confirmed our previous investigation on the response of E. faecalis to oxygen and again highlighted the apparent involvement of benzoic acid as a biomarker for aerobic growth in E. faecalis.