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Extracellular zinc competitively inhibits manganese uptake and compromises oxidative stress management in Streptococcus pneumoniae.

Eijkelkamp BA, Morey JR, Ween MP, Ong CL, McEwan AG, Paton JC, McDevitt CA - PLoS ONE (2014)

Bottom Line: We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway.Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor.Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a 'toxic' effect on bacterial pathogens, such as S. pneumoniae.

View Article: PubMed Central - PubMed

Affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, South Australia, Australia.

ABSTRACT
Streptococcus pneumoniae requires manganese for colonization of the human host, but the underlying molecular basis for this requirement has not been elucidated. Recently, it was shown that zinc could compromise manganese uptake and that zinc levels increased during infection by S. pneumoniae in all the niches that it colonized. Here we show, by quantitative means, that extracellular zinc acts in a dose dependent manner to competitively inhibit manganese uptake by S. pneumoniae, with an EC50 of 30.2 µM for zinc in cation-defined media. By exploiting the ability to directly manipulate S. pneumoniae accumulation of manganese, we analyzed the connection between manganese and superoxide dismutase (SodA), a primary source of protection for S. pneumoniae against oxidative stress. We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway. Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor. SodA was also shown to provide the majority of protection against oxidative stress as a S. pneumoniae ΔsodA mutant strain was found to be hypersensitive to oxidative stress, despite having wild-type manganese levels, indicating that the metal ion alone was not sufficiently protective. Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a 'toxic' effect on bacterial pathogens, such as S. pneumoniae.

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Competitive effect of Zn(II) on metal ion accumulation.(A) The concentration response curve fitting data for Mn(II) accumulation in S. pneumoniae D39 under extracellular Zn(II) stress. Data were normalized by comparison with non-competitive growth conditions [CDM + 1 µM Mn(II)]. Curve fitting was performed in Graphpad Prism version 5.0d (Graphpad). (B, C, D, and E) S. pneumoniae total cellular accumulation of Fe(II/III) (B), Co(II) (C), Ni(II) (D), and Cu(II) (E), determined by ICP-MS, when grown in CDM supplemented with 1 µM Mn(II), 10 µM Zn(II):1 µM Mn(II), 30 µM Zn(II):1 µM Mn(II), and 100 µM Zn:1 µM Mn. Data are mean (± SEM) µg metal.g dry cell mass−1 measurements from duplicate measurements of at least 3 independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired t-test (n.s. corresponds to not significant, * to P value < 0.05, and ** to P value < 0.01).
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pone-0089427-g002: Competitive effect of Zn(II) on metal ion accumulation.(A) The concentration response curve fitting data for Mn(II) accumulation in S. pneumoniae D39 under extracellular Zn(II) stress. Data were normalized by comparison with non-competitive growth conditions [CDM + 1 µM Mn(II)]. Curve fitting was performed in Graphpad Prism version 5.0d (Graphpad). (B, C, D, and E) S. pneumoniae total cellular accumulation of Fe(II/III) (B), Co(II) (C), Ni(II) (D), and Cu(II) (E), determined by ICP-MS, when grown in CDM supplemented with 1 µM Mn(II), 10 µM Zn(II):1 µM Mn(II), 30 µM Zn(II):1 µM Mn(II), and 100 µM Zn:1 µM Mn. Data are mean (± SEM) µg metal.g dry cell mass−1 measurements from duplicate measurements of at least 3 independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired t-test (n.s. corresponds to not significant, * to P value < 0.05, and ** to P value < 0.01).

Mentions: Analysis of the quantitative metal accumulation data for Mn(II) collected in this study revealed that Zn(II) had an EC50 for Mn(II) accumulation at a ratio of 30.2 µM Zn(II):1 µM Mn(II) in CDM (Fig. 2A). Therefore, the effect of extracellular Zn(II) on Mn(II) accumulation was consistent with competitive phenomena, which previously could only be inferred [26]. To assess whether extracellular Zn(II) affected the accumulation of other metal ions we further investigated the impact of competitive Zn(II) concentrations. As can be seen in Figures 2B-E, the effect of extracellular Zn(II) on metal accumulation was primarily restricted to Mn(II) accumulation, with no significant reduction in the accumulation of other transition row metals observed at any of the competitive ratios examined. However, Co(II) and Ni(II) did show minor increases at 10 µM Zn(II): 1 µM Mn(II), but as accumulation of these metal ions were not observed at other concentrations the significance of these changes were not readily apparent. Overall, these data indicate that the competitive effect of Zn(II) was primarily restricted to the Mn(II) uptake pathway. Collectively, these data provide direct quantitative evidence that extracellular Zn(II) competitively inhibits Mn(II) uptake in S. pneumoniae in a dose dependent manner and that the effect of Zn(II) primarily occurs on this pathway with negligible effects on other transition row metal ion transporter or the Zn(II) homeostatic mechanisms.


Extracellular zinc competitively inhibits manganese uptake and compromises oxidative stress management in Streptococcus pneumoniae.

Eijkelkamp BA, Morey JR, Ween MP, Ong CL, McEwan AG, Paton JC, McDevitt CA - PLoS ONE (2014)

Competitive effect of Zn(II) on metal ion accumulation.(A) The concentration response curve fitting data for Mn(II) accumulation in S. pneumoniae D39 under extracellular Zn(II) stress. Data were normalized by comparison with non-competitive growth conditions [CDM + 1 µM Mn(II)]. Curve fitting was performed in Graphpad Prism version 5.0d (Graphpad). (B, C, D, and E) S. pneumoniae total cellular accumulation of Fe(II/III) (B), Co(II) (C), Ni(II) (D), and Cu(II) (E), determined by ICP-MS, when grown in CDM supplemented with 1 µM Mn(II), 10 µM Zn(II):1 µM Mn(II), 30 µM Zn(II):1 µM Mn(II), and 100 µM Zn:1 µM Mn. Data are mean (± SEM) µg metal.g dry cell mass−1 measurements from duplicate measurements of at least 3 independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired t-test (n.s. corresponds to not significant, * to P value < 0.05, and ** to P value < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928430&req=5

pone-0089427-g002: Competitive effect of Zn(II) on metal ion accumulation.(A) The concentration response curve fitting data for Mn(II) accumulation in S. pneumoniae D39 under extracellular Zn(II) stress. Data were normalized by comparison with non-competitive growth conditions [CDM + 1 µM Mn(II)]. Curve fitting was performed in Graphpad Prism version 5.0d (Graphpad). (B, C, D, and E) S. pneumoniae total cellular accumulation of Fe(II/III) (B), Co(II) (C), Ni(II) (D), and Cu(II) (E), determined by ICP-MS, when grown in CDM supplemented with 1 µM Mn(II), 10 µM Zn(II):1 µM Mn(II), 30 µM Zn(II):1 µM Mn(II), and 100 µM Zn:1 µM Mn. Data are mean (± SEM) µg metal.g dry cell mass−1 measurements from duplicate measurements of at least 3 independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired t-test (n.s. corresponds to not significant, * to P value < 0.05, and ** to P value < 0.01).
Mentions: Analysis of the quantitative metal accumulation data for Mn(II) collected in this study revealed that Zn(II) had an EC50 for Mn(II) accumulation at a ratio of 30.2 µM Zn(II):1 µM Mn(II) in CDM (Fig. 2A). Therefore, the effect of extracellular Zn(II) on Mn(II) accumulation was consistent with competitive phenomena, which previously could only be inferred [26]. To assess whether extracellular Zn(II) affected the accumulation of other metal ions we further investigated the impact of competitive Zn(II) concentrations. As can be seen in Figures 2B-E, the effect of extracellular Zn(II) on metal accumulation was primarily restricted to Mn(II) accumulation, with no significant reduction in the accumulation of other transition row metals observed at any of the competitive ratios examined. However, Co(II) and Ni(II) did show minor increases at 10 µM Zn(II): 1 µM Mn(II), but as accumulation of these metal ions were not observed at other concentrations the significance of these changes were not readily apparent. Overall, these data indicate that the competitive effect of Zn(II) was primarily restricted to the Mn(II) uptake pathway. Collectively, these data provide direct quantitative evidence that extracellular Zn(II) competitively inhibits Mn(II) uptake in S. pneumoniae in a dose dependent manner and that the effect of Zn(II) primarily occurs on this pathway with negligible effects on other transition row metal ion transporter or the Zn(II) homeostatic mechanisms.

Bottom Line: We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway.Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor.Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a 'toxic' effect on bacterial pathogens, such as S. pneumoniae.

View Article: PubMed Central - PubMed

Affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, South Australia, Australia.

ABSTRACT
Streptococcus pneumoniae requires manganese for colonization of the human host, but the underlying molecular basis for this requirement has not been elucidated. Recently, it was shown that zinc could compromise manganese uptake and that zinc levels increased during infection by S. pneumoniae in all the niches that it colonized. Here we show, by quantitative means, that extracellular zinc acts in a dose dependent manner to competitively inhibit manganese uptake by S. pneumoniae, with an EC50 of 30.2 µM for zinc in cation-defined media. By exploiting the ability to directly manipulate S. pneumoniae accumulation of manganese, we analyzed the connection between manganese and superoxide dismutase (SodA), a primary source of protection for S. pneumoniae against oxidative stress. We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway. Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor. SodA was also shown to provide the majority of protection against oxidative stress as a S. pneumoniae ΔsodA mutant strain was found to be hypersensitive to oxidative stress, despite having wild-type manganese levels, indicating that the metal ion alone was not sufficiently protective. Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a 'toxic' effect on bacterial pathogens, such as S. pneumoniae.

Show MeSH
Related in: MedlinePlus