Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages

Abstract

The gene encoding the manganese-containing superoxide dismutase (MnSOD) of Enterococcus faecalis was characterized. It is transcribed monocistronically from an upstream promoter identified by rapid amplification of cDNA ends (RACE)-PCR. A sodA mutant was constructed and characterized. Growth of the mutant strain was not significantly different from that of its wild-type counterpart in standing and aerated cultures. However, the mutant was more sensitive towards menadione and hydroperoxide stresses. The response to H2O2 stress was analysed in more detail, and the mode of killing of this oxidant was different under anaerobic and aerobic conditions. Cultures grown and challenged under anaerobic conditions were highly sensitive to treatment with 35 mM H2O2. They were largely protected by the iron chelator deferoxamine, which suggested that killing was mainly due to an enhanced Fenton reaction. In contrast, neither strain was protected by the iron chelators deferoxamine and diethylenetriaminepentaacteic acid when grown and challenged under aerobic conditions, which suggested that inactivation of the cells by H2O2 was due to another killing mode. The sodA mutant was more sensitive under these conditions, showing that MnSOD is also important for protecting the cells from damage under aerobic conditions. Finally, the MnSOD of Ent. faecalis may be considered to be a virulence factor, since survival of the corresponding mutant strain was highly affected inside mouse peritoneal macrophages.

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

“…As mentioned above, the metabolism of glycerol via the GlpK pathway leads to the generation of H 2 O 2 , and we demonstrated that transient accumulation of this peroxide occurred during growth of the E. faecalis JH2-2 wild-type strain, leading to a growth inhibition which became even more pronounced in mutants affected in peroxidases (24). Since ⌬sodA mutants are more sensitive to externally added H 2 O 2 (18,30,31), the possibility that the growth on glycerol of the JH2-2 ⌬sodA mutant was affected by the intracellular generation of H 2 O 2 during catabolism of this substrate could not be excluded, and we tested this experimentally. While the wild-type JH2-2 strain grew to a final OD 600 of 3.5, the isogenic ⌬sodA mutant entered stationary phase early after start of growth and reached a final OD 600 of only 0.4 (see Fig.…”

The prolipoprotein diacylglyceryl transferase (Lgt) of Enterococcus faecalis contributes to virulence

“…4a). In E. faecalis, SOD has been shown to be important in protecting cells from damage under oxidative stress (Verneuil et al, 2006). However, zymographic analysis revealed no obvious change in the level of SOD activity in the Slgt mutant compared with the wild-type strain (data not shown).…”

Stress Physiology of Lactic Acid Bacteria

“…In contrast to streptococci, E. faecalis contains the heme-dependent catalase KatA, which is activated by heme taken up from the environment (259); its implication for virulence has not yet been investigated. Deletion of E. faecalis sodA attenuated resistance to H 2 O 2 and survival in mouse macrophages (254) and microglia (525). E. faecalis also contains three peroxidases: Npr, AhpCF, and Tpx (248).…”

“…An alternative strategy to counteract superoxide anion radicals is by the production of high levels of intracellular glutathione (252). Various enzymes that are known to counter oxidative stress, namely, MsrAB, AhpCF, Tpx, Npr, KatA, Sod, and HypR, have been characterized for E. faecalis (94,248,(253)(254)(255).…”