Impact of manganese, copper and zinc ions on the transcriptome of the nosocomial pathogen Enterococcus faecalis V583

Abstract

Mechanisms that enable Enterococcus to cope with different environmental stresses and their contribution to the switch from commensalism to pathogenicity of this organism are still poorly understood. Maintenance of intracellular homeostasis of metal ions is crucial for survival of these bacteria. In particular Zn(2+), Mn(2+) and Cu(2+) are very important metal ions as they are co-factors of many enzymes, are involved in oxidative stress defense and have a role in the immune system of the host. Their concentrations inside the human body vary hugely, which makes it imperative for Enterococcus to fine-tune metal ion homeostasis in order to survive inside the host and colonize it. Little is known about metal regulation in Enterococcus faecalis. Here we present the first genome-wide description of gene expression of E. faecalis V583 growing in the presence of high concentrations of zinc, manganese or copper ions. The DNA microarray experiments revealed that mostly transporters are involved in the responses of E. faecalis to prolonged exposure to high metal concentrations although genes involved in cellular processes, in energy and amino acid metabolisms and genes related to the cell envelope also seem to play important roles.

Figure 1. E. faecalis V583 growth in GM17 with and without added metals.

A. E. faecalis V583 growth in GM17 (empty diamonds) and GM17 with 4 mM ZnSO₄ (empty squares), 5 mM ZnSO₄ (empty triangles) or 6 mM ZnSO₄ (crosses); B. E. faecalis V583 growth in GM17 (empty diamonds) and GM17 with 0.4 mM MnSO₄ (empty triangles), 1 mM MnSO₄ (crosses) or 2 mM MnSO₄ (asterisks); C. E. faecalis V583 growth in GM17 (empty diamonds) and GM17 with 0.05 mM CuSO₄ (empty squares), 0.075 mM CuSO₄ (empty triangles) and 0.100 mM CuSO₄ (crosses). Indicated are the added metal concentrations. All experiments were performed in triplicate.

Figure 2. Representation of E. faecalis V583 transcriptome responses to metal stress by functional categories.

Representation of the weight of each functional category in the transcriptome responses of E. faecalis V583 to high concentrations of Zn²⁺, Mn²⁺ or Cu²⁺. The weight represents the percentage of genes differentially expressed in each category relative to the total number of predicted genes in that category in the genome of V583. Zinc experiments – black/grey bars; manganese experiments - dark pink/light pink; copper experiments – dark blue/light blue. The weight in percentage is given at the sides of the bars.

Figure 3. Distribution of differentially expressed genes in the presence of high concentrations of metals.

Distribution of E. faecalis V583 genes differentially expressed in the presence of 4 mM Zn²⁺ (Zn), 0.4 mM Mn²⁺ (Mn) and 0.05 mM Cu²⁺ (Cu). Venn diagram showing the number of unique and common differentially expressed genes in the three metal DNA microarray experiments.

Figure 4. Heat map of differentially expressed genes grown in blood, in urine and with metal stresses.

a) Heat map visualizing the regulated genes in the trancriptome experiments with blood , urine and zinc, manganese and copper stresses (this work). b) Detail of the heat map showing the transcriptome results for the efaCBA operon. Genes found to be significantly regulated are indicated by either red (up-regulated) or green (down-regulated).