EfaR is a major regulator of Enterococcus faecalis manganese transporters and influences processes involved in host colonization and infection

Abstract

Metal ions, in particular manganese, are important modulators of bacterial pathogenicity. However, little is known about the role of manganese-dependent proteins in the nosocomial pathogen Enterococcus faecalis, a major cause of bacterial endocarditis. The present study demonstrates that the DtxR/MntR family metalloregulator EfaR of E. faecalis controls the expression of several of its regulon members in a manganese-dependent way. We also show that efaR inactivation impairs the ability of E. faecalis to form biofilms, to survive inside macrophages, and to tolerate oxidative stress. Our results reveal that EfaR is an important modulator of E. faecalis virulence and link manganese homeostasis to enterococcal pathogenicity.

Fig 1

The EBM, present in the promoter regions of several genes differentially expressed in the presence of excess Zn²⁺, Mn²⁺, or Cu²⁺ ions. The WebLogo is based on the MEME weight matrix and shows the bit score of A, C, T, and G nucleotides at each position of the motif (44).

Fig 2

Effect of metal addition on E. faecalis. All strains carry plasmid pSAVE11, which contains a fusion of E. coli lacZ to the EBM-containing promoter of mntH2. (A) lacZ expression in wild-type VE14089 (pSAVE11) grown in GM17 with the indicated added concentrations of ZnSO₄, MnCl₂, CuSO₄, or FeCl₂. (B) lacZ expression in the wild type (pSAVE11) and the efaR mutant (pSAVE11) grown in GM17 with ZnSO₄ (0 mM or 4 mM), MnCl₂ (0 mM or 0.4 mM), or CuSO₄ (0 mM or 0.05 mM). *, P < 0.03.

Fig 3

Effect of metal addition on E. faecalis lacZ expression in the wild-type strain VE14089 and its isogenic efaR mutant, both carrying pSAVE11 with the P_mntH2::lacZ fusion, under metal-depleted conditions. Values are lacZ expression in the strains grown in chelGM17 with ZnSO₄ (0 mM or 4 mM), MnCl₂ (0 mM or 0.4 mM), or CuSO₄ (0 mM or 0.05 mM). *, P < 0.03.

Fig 4

Effect of metal addition in wild-type E. faecalis VE14089 or its ef0579 mutant, each carrying pSAVE10, a plasmid with a Pef0575::lacZ fusion, or pSAVE11, a plasmid with a PmntH2::lacZ fusion. (A) lacZ expression in VE14089 (pSAVE10) and the ef0579 mutant (pSAVE10) grown in GM17 with ZnSO₄ (Zn; 0 and 4 mM), MnCl₂ (Mn; 0 and 0.4 mM), or CuSO₄ (Cu; 0 and 0.05 mM); (B) lacZ expression in VE14089 (pSAVE11) and the ef0579 mutant (pSAVE11) grown in GM17 with ZnSO₄ (Zn; 0 and 4 mM), MnCl₂ (Mn; 0 and 0.4 mM), or CuSO₄ (Cu; 0 and 0.05 mM). *, P < 0.03.

Fig 5

Intramacrophage survival of E. faecalis. Percentage of survival of E. faecalis VE14089 (gray bars) and the efaR mutant (black bars) inside macrophages (J774 A1 cells) at the indicated times after addition of the bacteria to a confluent layer of macrophages. Bacterial survival was measured by plating on BHI agar plates. Results are the means from three independent experiments. *, P < 0.03, calculated in comparison with the value at time zero.

Fig 6

E. faecalis tolerance to oxidative stress. Survival of E. faecalis VE14089 (diamonds) and its isogenic efaA, efaR, and ef0577 insertion mutants at 15, 30, and 60 min after challenge with 20 mM H₂O₂. CFU were determined by plate counting using BHI plates. A value of 100% corresponds to the number of CFU immediately prior to the additions of H₂O₂ (0 min). The values are the averages from three independent experiments. *, P < 0.03, calculated in comparison with the value at time zero.

Fig 7

E. faecalis biofilm formation. The biofilm-forming ability of wild-type E. faecalis VE14089 and its efaR, efaA, and ef0577 mutants was quantified using crystal violet staining (34). Values are averages of results obtained in three independent experiments, performed in six replicates. *, P < 0.03.