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Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections.

Cho H, Jeong DW, Liu Q, Yeo WS, Vogl T, Skaar EP, Chazin WJ, Bae T - PLoS Pathog. (2015)

Bottom Line: The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression.During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality.These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.

View Article: PubMed Central - PubMed

Affiliation: Indiana University School of Medicine-Northwest, Gary, Indiana, United States of America.

ABSTRACT
Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.

No MeSH data available.


Related in: MedlinePlus

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.
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ppat.1005026.g003: 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.

Mentions: Since the SaeRS TCS is activated by neutrophils [35], in the following studies, we focused our investigation on Ca, Mn, Fe, and Zn, the major divalent ions found in neutrophil granules [32]. We reasoned that, since CP binds Zn with high affinity, it might be able to reduce the Zn-mediated repression of the SaeRS TCS. To test this possibility, we grew S. aureus cells in RPMI supplemented with CP and one or all the four metal ions and measured P1promoter activity and SaeQ expression. When the growth medium was supplemented with Fe, as expected, CP failed to restore the activity of the SaeRS TCS (Fig 3A). However, when the medium was supplemented with Zn, CP restored the activity of the SaeRS TCS (Fig 3A), demonstrating that indeed CP can protect the SaeRS TCS activity from the Zn-mediated repression. Intriguingly, when the growth medium was supplemented with all four metal ions, CP restored the SaeRS TCS activity, despite the presence of Fe (Fig 3A). We noted that, in the experiment above, we added 364 times more Zn (400 μM) than CP (1.1 μM) to the growth medium. Therefore, most of Zn ions (> 99%) are expected not to be bound to CP, and simple sequestration of Zn by CP cannot explain the restoration of the SaeRS TCS activity. In addition, despite the fact that CP does not bind to Fe, when CP was added to the growth medium supplemented with all the four metal ions, it restored SaeRS TCS activity (Fig 3A). Based on these results, we hypothesized that, by binding to Zn, CP gains the ability to protect the SaeRS TCS not only from Zn but also from Fe. To test this hypothesis, we grew the strain USA300 in RPMI, added Fe and Zn-CP mixture of various ratios (0–364), and then measured the SaeRS TCS activity. Indeed, CP restored the SaeQ expression only when Zn was also present (Fig 3B), and the restoration of SaeQ expression required more than 2 h incubation (Fig 3C). Moreover, a mutant CP incapable of binding to Zn failed to protect the activity of the SaeRS TCS, while CP mutants retaining one binding site still protected [20] (Fig 3D). These results suggest that binding of Zn confers CP with the ability to protect the SaeRS TCS from repression by Zn and Fe.


Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections.

Cho H, Jeong DW, Liu Q, Yeo WS, Vogl T, Skaar EP, Chazin WJ, Bae T - PLoS Pathog. (2015)

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.
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ppat.1005026.g003: 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.
Mentions: Since the SaeRS TCS is activated by neutrophils [35], in the following studies, we focused our investigation on Ca, Mn, Fe, and Zn, the major divalent ions found in neutrophil granules [32]. We reasoned that, since CP binds Zn with high affinity, it might be able to reduce the Zn-mediated repression of the SaeRS TCS. To test this possibility, we grew S. aureus cells in RPMI supplemented with CP and one or all the four metal ions and measured P1promoter activity and SaeQ expression. When the growth medium was supplemented with Fe, as expected, CP failed to restore the activity of the SaeRS TCS (Fig 3A). However, when the medium was supplemented with Zn, CP restored the activity of the SaeRS TCS (Fig 3A), demonstrating that indeed CP can protect the SaeRS TCS activity from the Zn-mediated repression. Intriguingly, when the growth medium was supplemented with all four metal ions, CP restored the SaeRS TCS activity, despite the presence of Fe (Fig 3A). We noted that, in the experiment above, we added 364 times more Zn (400 μM) than CP (1.1 μM) to the growth medium. Therefore, most of Zn ions (> 99%) are expected not to be bound to CP, and simple sequestration of Zn by CP cannot explain the restoration of the SaeRS TCS activity. In addition, despite the fact that CP does not bind to Fe, when CP was added to the growth medium supplemented with all the four metal ions, it restored SaeRS TCS activity (Fig 3A). Based on these results, we hypothesized that, by binding to Zn, CP gains the ability to protect the SaeRS TCS not only from Zn but also from Fe. To test this hypothesis, we grew the strain USA300 in RPMI, added Fe and Zn-CP mixture of various ratios (0–364), and then measured the SaeRS TCS activity. Indeed, CP restored the SaeQ expression only when Zn was also present (Fig 3B), and the restoration of SaeQ expression required more than 2 h incubation (Fig 3C). Moreover, a mutant CP incapable of binding to Zn failed to protect the activity of the SaeRS TCS, while CP mutants retaining one binding site still protected [20] (Fig 3D). These results suggest that binding of Zn confers CP with the ability to protect the SaeRS TCS from repression by Zn and Fe.

Bottom Line: The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression.During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality.These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.

View Article: PubMed Central - PubMed

Affiliation: Indiana University School of Medicine-Northwest, Gary, Indiana, United States of America.

ABSTRACT
Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.

No MeSH data available.


Related in: MedlinePlus