Fig 1.
The SaeRS TCS can be repressed by Cu, Fe, and Zn.
(A) The effect of culture medium on the expression of SaeQ. S. aureus cells were grown to 0.5 OD600, and SaeQ protein was detected by Western blot analysis. Newman, S. aureus strain Newman; USA300, S. aureus strain USA300; + EDTA, addition of 1 mM EDTA. The quantification result of the Western blot is shown to the right. (B) The effect of iron on the P1 promoter activity (top) and SaeQ expression (bottom). P1 promoter activity was measured by LacZ assay for P1-lacZ fusion construct, while SaeQ was measured by Western blot analysis. -, no metal addition. (C) The effect of metal ions present in human blood on the P1 promoter activity (top) and SaeQ expression (bottom). S. aureus cells were grown in RPMI supplemented with 2 mM CaCl2, 20 μM CuSO4, 20 μM FeSO4, 500 μM MgSO4, 0.1 μM MnSO4, 0.04 μM NiSO4, or 20 μM ZnSO4 at 37°C for 16 h. Then the P1 promoter activity and SaeQ expression were measured by LacZ assay. Statistical comparison was made against the no metal (-) condition. (D) The effect of metal ions present in human neutrophil granules on the P1 promoter activity and SaeQ expression. Cells were grown in RPMI supplemented with 400 μM CaCl2, 130 μM FeSO4, 130 μM MnSO4, or 400 μM ZnSO4. All other conditions are the same as in (C). (E) The effect of metal chelation on the recovery of the P1 promoter activity (top) and SaeQ expression (bottom). S. aureus cells were grown for 16 h in RPMI supplemented with all metal ions (Mix) used in (D). The presented data represent three independent experiments. Error bars indicate standard error of the mean. Statistical significance was measured by unpaired, two-tailed t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001
Fig 2.
Cu and Zn inhibit the autokinase activity of SaeS.
(A) The effect of Fe, Zn and Cu on the autokinase activity of SaeS. MBP-SaeS (3 μM) was autophosphorylated with [γ-32P]-ATP for 15 min in the presence of various concentrations of FeSO4, ZnSO4, and CuSO4. Then the phosphorylation of SaeS was measured by SDS-PAGE and autoradiograph (top) and quantified (bottom). (B) The effect of Zn on the autokinase activity of SaeS in purified cell membranes. Cell membranes (25 μg) containing overexpressed SaeS were used as a source of SaeS. The experiment was carried out as described above. The autoradiograph (top) and quantification of the results (bottom) are shown.
Fig 3.
Calprotectin protects the SaeRS TCS from the metal-mediated repression.
(A) Effect of calprotectin (CP) on the P1 promoter activity and SaeQ expression in the presence of metals found in neutrophil granules. S. aureus was grown for 16 h in RPMI supplemented with CP (1.1 μM) and the following metal: 400 μM CaCl2, 130 μM FeSO4, 130 μM MnSO4, or 400 μM ZnSO4. -, no metal addition; +, addition. (B) Effect of Zn-CP on Fe-mediated repression of the SaeRS TCS. Cells were grown in RPMI to exponential growth phase; then FeSO4 (130 μM), CP (1.1 μM), and various concentrations of ZnSO4 were added. The cells were further incubated for 16 h. Zn/CP, molar ratio of Zn and CP. (C) Effect of incubation time on the restoration of SaeQ expression. Cells were prepared as described above (B) and collected at 2 h and 4 h post incubation. The level of SaeQ was measured by Western blot analyses. (D) The effect of Zn-binding on the CP’s protective effect on the SaeRS TCS. Cells were grown to exponential growth phase; then FeSO4 (130 μM), ZnSO4 (22 μM) and wild type or Zn-binding mutants of CP (1.1 μM) were added. The cells were further incubated for 16 h. Δ1, a mutant CP where the Zn/Mn binding site S1 was abolished; Δ2, a mutant CP where the Zn binding site S2 was abolished; Δ1Δ2, a mutant CP where both S1 and S2 binding sites were abolished. (E) The effect of Zn-CP complex on the Fe-mediated repression of the SaeRS TCS in the presence of HNP1. Cells were grown in RPMI to exponential growth phase; then HNP1 (1.5 μM), FeSO4 (130 μM), CP (1.1 μM) and ZnSO4 (22 μM) were added in the various combinations indicated. The P1 promoter activity (top) and the SaeQ expression (bottom) were measured at 16 h post incubation. Statistical significance was measured by unpaired, two-tailed t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 4.
RNA-seq analysis of the effect of CP, Zn and Zn-CP on staphylococcal transcriptome.
S. aureus USA300 cells at exponential growth phase were treated by CP (1.1 μM), Zn (20 μM) or Zn (20 μM)-CP (1.1 μM) at 37°C for 4 h; then the transcript levels were analyzed by RNA-seq. (A) The number of genes affected by CP, Zn, and Zn-CP. (B) Venn diagram analysis of the genes affected by CP, Zn, and Zn-CP. Up, up-regulated genes; Down, down-regulated genes. (C) Effect of CP, Zn, and Zn-CP on the transcript levels of the sae regulon. -, no treatment. ID, Gene ID in the genome of the strain USA300_FPR3757.
Fig 5.
CP is required for full activation of the SaeRS TCS and cytokine production by murine neutrophils.
(A) Activation of the SaeRS TCS by neutrophils from C57BL/6 (WT) and C57BL/6 S100A9 -/- (A9-/-) mice. Neutrophils purified from bone marrow of WT or A9-/- mice were mixed with S. aureus strain USA300 containing P1-gfp reporter plasmid (MOI = 10). At the indicated time points, neutrophils were lysed, and the P1 promoter activity was measured by flow cytometry (top panel), and the results were also presented in a bar graph (bottom panel). In the flow cytometry analysis, gray color represents the results from the control plasmid (pCL-gfp). In the bar graph, error bars depict standard error of the mean. Results are from three pooled mice per genotype and represent three independent experiments. (B) The effect of CP in the cytokine production of neutrophil. Neutrophils purified from bone marrow of WT and S100A9-/- mice were infected with S. aureus USA300 (MOI = 10) for 2 h. After addition of gentamicin, neutrophils were further incubated for 16 h, and the concentration of the cytokines indicated was determined by ELISA. The data are from three pooled animals per genotype and representative of three independent experiments. Error bars indicate standard error of the mean. Statistical significance was determined by unpaired, two tailed t-test.
Fig 6.
CP enhances the activity of the SaeRS TCS and cytokine production in mice.
(A) The activity of the SaeRS TCS in C57BL/6 (WT) and C57BL/6 S100A9-/- (A9-/-) mice. Mice were infected with S. aureus (2 × 108 cfu) by peritoneal injection, and peritoneal fluid was acquired at the indicated time points. After being separated by centrifugation (200 ×g), the GFP expression of S. aureus in supernatant (host cell-free) or in pellet (host cell-associated) was measured by flow cytometry (top panel), where the gray color represents GFP expression from pCL-gfp. The quantification results are shown in S5 Fig. (B) The effect of CP on the expression of select genes. (C) The production of proinflammatory cytokines in wild type (WT) and S100A9-/- (A9-/-) mice upon infection with S. aureus USA300 (USA) or the sae deletion mutant (Δsae). At the time points indicated, mice were sacrificed and the concentrations of proinflammatory cytokines in peritoneal fluids were determined by ELISA. The data are from three pooled mice per genotype and represent three independent experiments. Error bars indicate standard error of the mean. -, no bacteria. Statistical significance was assessed by t-test. * p < 0.05; ** p < 0.01; *** p < 0.001
Fig 7.
The proinflammatory property of CP increases murine mortality.
(A) Effect of CP on the survival of mice infected by S. aureus. C57BL/6 (WT) or C57BL/6 S100A9-/- (A9-/-) mice were infected with USA300 (USA) or sae-deletion mutant (Δsae) by intraperitoneal injection (2×108 cfu). Ten mice were used for each test group. Statistical significance was assessed by Log-rank (Mantel-Cox) test. (B) Effect of individual proinflammatory cytokine antibodies on the murine survival. WT or A9-/- mice were infected with the wild type USA300 (2 × 108 cfu). At 2 h post infection, 100 μg of the indicated antibody was injected via i.p. route. (C) Effect of proinflammatory cytokine antibody mixture on the murine survival. WT or A9-/- mice were infected with the wild type USA300 (2 × 108 cfu). At 2 h post infection, 150 μg of proinflammatory cytokine antibody (50 μg of each IFN-γ Ab, TNF-α Ab, and IL-1β Ab) was injected via i.p. route. (D) CP can increase murine mortality in the absence of the SaeRS TCS. WT or A9-/- mice were infected with the sae-deletion mutant (Δsae, 1 × 109 cfu). Statistical significance was assessed by Log-rank (Mantel-Cox) test. ns, not significant; **, p < 0.01; ***, p < 0.001