pmc.ncbi.nlm.nih.gov

Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense enhancing neutrophil killing of Staphylococcus aureus

. Author manuscript; available in PMC: 2012 Aug 18.

Published in final edited form as: Cell Host Microbe. 2011 Aug 18;10(2):158–164. doi: 10.1016/j.chom.2011.07.004

Summary

By sequestering manganese and zinc, the neutrophil protein calprotectin plays a crucial role in host defense against bacterial and fungal pathogens. However, the essential processes disrupted by calprotectin remain unknown. We report that calprotectin enhances the sensitivity of Staphylococcus aureus to superoxide through inhibition of manganese-dependent bacterial superoxide defenses thereby increasing superoxide levels within the bacterial cell. Superoxide dismutase activity is required for full virulence in a systemic model of S. aureus infection and disruption of staphylococcal superoxide defenses by calprotectin augments the antimicrobial activity of neutrophils promoting in vivo clearance. Calprotectin mutated in two transition metal binding sites and therefore defective in binding manganese and zinc does not inhibit microbial growth, unequivocally linking the antimicrobial properties of calprotectin to metal chelation. These results suggest that calprotectin contributes to host defense by rendering bacterial pathogens more sensitive to host immune effectors and reducing bacterial growth.

Introduction

Staphylococcus aureus is a bacterial pathogen of substantial concern due to the emergence of antibiotic resistant strains and the bacterium’s ability to infect nearly every organ (Grundmann et al., 2006; Lowy, 1998; Said-Salim et al., 2003). For pathogens such as S. aureus to survive and replicate within a vertebrate host they must acquire essential nutrients from the host environment (Andreini et al., 2008; Corbin et al., 2008; Kehl-Fie and Skaar, 2010). To combat invading pathogens the vertebrate host exploits the requirement for nutrient metals by limiting their availability, a process termed “nutritional immunity” (Weinberg, 2009). In a mouse model of S. aureus infection it has been shown that vertebrates reduce Mn and Zn levels within tissue abscesses to nearly undetectable levels (Corbin et al., 2008). The neutrophil protein calprotectin (CP) binds these metals and is essential for the sequestration of Mn during infection (Corbin et al., 2008; Yousefi et al., 2007).

CP is a member of the S100 class of EF-hand calcium (Ca) binding proteins composed of the heterodimeric complex of S100A8 and S100A9. CP comprises approximately 50% of the proteinaceous content of the neutrophil cytoplasm leading to concentrations in excess of 1 mg/ml within tissue abscesses (Clohessy and Golden, 1995; Gebhardt et al., 2006). While Mn and Zn sequestration by CP inhibits microbial growth and contributes to the control of pathogens, the essential processes that are disrupted remain unknown (Corbin et al., 2008; Urban et al., 2009). One potential target of CP-mediated metal deprivation is Mn-dependent superoxide defenses, which protect invading microbes from the oxidative burst of neutrophils (Babior, 2004; Lynch and Kuramitsu, 2000).

The studies reported here investigated the ability of CP to inhibit staphylococcal Mn-dependent superoxide defense. We found that CP reduces the activity of Mn-dependent superoxide dismutases (SOD) as well as Mn-dependent SOD-independent mechanisms of superoxide defense. These activities correlate with the binding affinities of CP for Mn2+ and Zn2+. Inactivation of these systems by CP increases intracellular superoxide and is dependent on CP-mediated metal chelation. Moreover, inactivation of SOD activity by CP renders S. aureus more sensitive to neutrophil-mediated killing and reduces virulence in a systemic model of infection.

Results

Calprotectin enhances the sensitivity of S. aureus to superoxide stress

S. aureus possesses two distinct Mn-requiring mechanisms for dealing with superoxide stress. The first mechanism is the dismutation of superoxide by SODs. S. aureus expresses two SODs, SodA and SodM, both of which require Mn for function (Clements et al., 1999; Valderas and Hart, 2001). The second mechanism protects against superoxide through as-yet-unidentified processes that also require Mn (Horsburgh et al., 2002a; Horsburgh et al., 2002b). To test the hypothesis that binding of Mn2+ by CP inhibits these processes, we assessed the effect of CP-treatment on the sensitivity of S. aureus to superoxide. Increasing concentrations of CP enhance the susceptibility of S. aureus strain Newman to the superoxide generators paraquat and xanthine/xanthine oxidase (Fig. 1A, 1B & S1A). A similar effect was observed for the community acquired methicillin resistant S. aureus strain USA300 (Fig. 1C & S1B) and the S. epidermidis strain ATCC 12228 (Fig. S1C & D), which possesses a single Mn-dependent SOD (Diep et al., 2006; Valderas et al., 2002). The addition of excess Mn2+ or Zn2+ protected S. aureus against superoxide stress in the presence of CP, implicating CP-mediated metal binding as contributing to this process (Fig. 1B, 1D, S1E & F). Growth of S. aureus Newman in the presence of CP and the superoxide scavenger glutathione protected S. aureus from the effects of paraquat, but did not entirely reverse the antimicrobial effects of CP (Fig 1E).

Figure 1. Calprotectin enhances the effects of superoxide stress.

Figure 1

A) Growth of S. aureus Newman in the presence of CP and/or paraquat. B) Survival of stationary phase S. aureus Newman or a sodA::tet sodM::erm (sodAsodM) exposed to either 1.5U (Newman) or 0.15U (sodAsodM) xanthine oxidase and 2 mM xanthine following growth in the presence of calprotectin or manganese. C) Growth of S. aureus USA300 in the presence of CP and/or paraquat. D) Growth of Newman or USA300 in the presence of 500 mM MnCl2, 240 mg/ml CP, and/or 1 mM paraquat. E) Growth of Newman in the presence of 500 mM MnCl2, 240 mg/ml CP, 1 mM paraquat and or the superoxide scavenging compound glutathione. F) Growth of a sodAsodM derivative of Newman in the presence of CP and/or paraquat. G) Growth of a sodAsodM derivative of Newman in the presence of 500 mM MnCl2, 240 mg/ml CP, and/or 0.1 mM paraquat. Panels A, B, C and F asterisks indicate p value less than 0.05 by one way ANOVA with Dunnet’s posttest. Panels D, E, and F asterisks indicate p value less than 0.05 by one way ANOVA with Bonferroni posttest of selected means. Means represent the average of at least three independent experiments performed in triplicate. Error bars = SD, CP = calprotectin, PQ = paraquat, Mn = MnCl2. (See also Figure S1)

Mn/Zn binding contributes to the antimicrobial activity of CP

Isothermal titration calorimetry (ITC) experiments on wild type (WT) CP revealed a stoichiometry of two Zn2+ ions with dissociation constants (Kd) of 1.35 nM and 5.6 nM, whereas only one Mn2+ ion was bound with high affinity (Kd 1.3 nM) (Fig. 2A). Binding of a second Mn2+ ion was observed, but with ~1000-fold weaker affinity (Kd 3.7 µM). In order to test the contribution of nutrient metal binding to the antimicrobial activity of CP, a mutant was designed to inactivate the predicted metal binding sites (ΔZn/Mn) based on a homology model of Zn-bound S100A8/S100A9 (Fig. S2). This model predicts the presence of one Mn/Zn binding site involving H17 and H27 from S100A8 and H91 and H95 from S100A9 (Site I, Fig. S2B). The second site involves H20 and D30 from S100A9 and H83 and H87 from S100A8 (Site II, Fig. S2B). The ΔZn/Mn CP mutant was generated by substituting each histidine for asparagine and the aspartic acid to serine, resulting in two constructs: S100A8 H17N/H27N/H83N/H87N, and S100A9 H20N/D30S/H91N/H95N. Comparison of two-dimensional 15N-1H HSQC NMR spectra of wild-type and ΔZn/Mn CP confirmed that the mutations had no significant effect on the secondary or tertiary structure (Fig. S2C & D). The affinity of ΔZn/Mn for Zn2+ and Mn2+ was then assayed by ITC and no significant binding of either ion was observed (Fig. 2B). Loss of Zn2+ and Mn2+ binding to ΔZn/Mn CP implies that the eight mutated residues are responsible for binding of these important nutritional metals in the wild type protein. To evaluate the contribution of metal chelation to the antimicrobial activity of CP, the IC50 of WT CP and ΔZn/Mn were compared. ΔZn/Mn has an IC50 of 9042 µg/ml (+/− 1568 SD) or approximately 60 times that of WT CP which has an IC50 of 139 µg/ml (+/− 9 SD) (Fig. S2E). The IC50 for ΔZn/Mn is ~9-fold higher than the concentration found within abscessed tissues, supporting the notion that a metal binding defective version of CP would not be antimicrobial in vivo (Clohessy and Golden, 1995). Additionally, ΔZn/Mn CP does not increase the sensitivity of S. aureus to superoxide stress (Fig. 2C & S2F). These data unequivocally link the metal chelating properties of CP to its antimicrobial activities.

Figure 2. Mn and Zn binding are necessary for the antimicrobial activity of calprotectin.

Figure 2

A & B) ITC thermograms for Mn2+ and Zn2+ binding to WT (A) and ΔZn/Mn (B) CP. NB = no detectable binding. C) Effect of ΔZn/Mn CP mutation on the sensitivity of S. aureus to superoxide stress. Newman was grown in the presence of ΔZn/Mn CP and/or paraquat. Growth was assessed by measuring OD600. Means represent the average of three independent experiments performed in triplicate. Error bars = SD, NS = not significant via one-way ANOVA with Dunnet’s posttest. (See also Figure S2)

CP increases superoxide levels and decreases SOD activity

To test if CP-mediated metal chelation increases the amount of superoxide stress experienced by S. aureus, we assessed intracellular superoxide levels using the membrane permeable dye dihydroethidium (DHE) (Carter et al., 1994). Increased levels of intracellular superoxide were observed when S. aureus was grown in the presence of 240 µg/ml CP (Fig. 3A), the same concentration of CP that increases the sensitivity of S. aureus to exogenous superoxide stress (Fig. 1A). To determine if CP treatment reduces staphylococcal SOD activity, a water-soluble tetrazolium salt (WST) assay was employed. Growth of S. aureus in the presence of CP resulted in decreased levels of SOD activity in both exponential and stationary phase bacteria as well as upon exposure to superoxide stress (Fig. 3B & S3A). The CP-mediated reduction in SOD activity is dependent on the metal chelating ability of CP as the addition of excess Mn2+ restores enzymatic activity, and ΔZn/Mn does not reduce S. aureus SOD activity (Fig. 3B). Finally, incubation of CP with purified SodA or SodM modestly reduced SOD activity (Fig. S3C, D & E). Overall, these data support the proposal that CP increases the superoxide sensitivity of S. aureus by reducing Mn2+ levels and preventing the formation of SOD-Mn complexes.

Figure 3. Calprotectin treatment results in reduced staphylococcal SOD activity and increased intracellular levels of superoxide.

Figure 3

A) Assessment of intracellular superoxide in S. aureus Newman or a sodA::tet sodM::erm (sodAsodM) derivative in increasing concentrations of CP as determined by a DHE assay. * = p < 0.05 one-way ANOVA with Dunnet’s posttest. EtBr = ethidium bromide. B) S. aureus Newman was treated with, buffer (No CP), WT CP or the ΔZn/Mn mutant, in the presence of Mn2+, and/or paraquat, and then grown to mid-exponential phase. SOD activity was assessed by water-soluble tetrazolium salt assay. Means represent the average of at least three independent experiments assayed in triplicate. Error bars = SD, * = p < 0.05 by one-way ANOVA with Bonferroni posttest of selected means. CP = calprotectin, Mn=MnCl2, PQ= paraquat, NS = not significant. (See also Figure S3)

SOD-independent mechanisms of superoxide defense are inhibited by CP

To determine if SOD-independent superoxide defenses are inhibited by CP, a sodA::tet sodM::erm (sodAsodM) mutant of S. aureus Newman was examined. The sodAsodM mutant is more sensitive to superoxide than Newman in the absence of CP treatment, and when treated with CP displays a further increase in sensitivity to superoxide stress that is reversed by Mn (Fig. 1B, 1F, 1G & S1G). Additionally, sodAsodM is approximately twice as sensitive to CP treatment as wild type Newman even in the absence of superoxide stress (Fig. S1H & I). sodAsodM exhibits only background levels of SOD activity indicating that CP is not inhibiting an unidentified third SOD in S. aureus (Fig. S3A & B). sodAsodM was also examined by DHE assay and found to have increased levels of intracellular superoxide upon exposure to as little as 60 µg/ml CP (Fig. 3A). These results indicate that CP inhibits both SOD-independent and SOD-dependent mechanisms of superoxide resistance.

Staphylococcal SODs are inhibited by CP during systemic infection

To ascertain whether CP-dependent inhibition of bacterial superoxide defenses augments the antibacterial activity of neutrophils, CP treated S. aureus was examined for sensitivity to neutrophil-mediated killing. CP enhanced the sensitivity of exponential phase S. aureus Newman but not sodAsodM to neutrophil-mediated killing (Fig. 4A). As staphylococcal SOD mutants become more resistant to superoxide stress in stationary phase (Karavolos et al., 2003), the impact of CP on the sensitivity of stationary phase bacteria was examined. In this growth condition, CP enhanced the sensitivity of both wild type S. aureus and the sodAsodM mutant to neutrophil-mediated killing (Fig 4B).

Figure 4. SODs contribute to systemic infection but can be inhibited by calprotectin rendering S. aureus more sensitive to neutrophil-mediated killing.

Figure 4

A & B) Casein elicited PMNs were incubated with S. aureus grown to exponential (A) or stationary (B) phase in the presence or absence of CP (750 µg/ml Newman or 450 µg/ml sodAsodM) and bacterial viability was determined by plating serial dilutions on solid medium. Data represent the mean of four or more independent experiments performed in triplicate. *= p < 0.05 globally via 2-way ANOVA and # = p <0.05 for comparison of specific time points via Bonferroni posttest. Error bars = SEM. C & D) Six-week old C57BL/6 (C) or 8–9 week old C57BL/6 (C57) and S100A9−/− C57BL/6 (A9−/−) mice (D) were infected with either ~1×107 S. aureus Newman or the sodAsodM mutant. Mice were sacrificed 96 hours following infection and bacterial loads in the livers were enumerated. Bars represent the mean of each infection and boxes represent standard deviation. The number of mice in each group is indicated by n = equals. *= p < 0.05 as determined by Students two tailed t-test.

Next, sodAsodM was examined for its ability to cause disease in a murine model of infection using 6 week old C57BL/6 mice. The sodAsodM mutant exhibited a 2-log reduction in bacterial load in the livers as compared to wild type bacteria (Fig. 4C). To test the impact of CP-mediated SOD inactivation during systemic S. aureus infections, 8–9 week old C57BL/6 and CP-deficient mice were infected with S. aureus wild type or the sodAsodM mutant (Fig 4D). Mice lacking CP are more susceptible to wild type S. aureus infection as demonstrated by an approximately 1-log increase in cfus in the livers of CP-deficient mice as compared to C57BL/6. Notably, the infectivity of the sodAsodM mutant did not differ significantly between C57BL/6 mice and CP-deficient mice. Taken together, these data suggest that calprotectin-mediated Mn2+ chelation reduces the activity of S. aureus SODs during infection, protecting the host against staphylococcal challenge.

Discussion

Vertebrates combat invading pathogens through the sequestration of essential nutrients in a process termed “nutritional immunity” (Weinberg, 2009). Sequestration of Mn is dependent on the neutrophil protein CP, which results in the inhibition of microbial growth and protection against infection (Corbin et al., 2008; Urban et al., 2009). While the antimicrobial activity of CP has been suggested to be dependent on nutrient metal chelation, the effect that this sequestration has on bacterial processes remains unclear. Based on the results reported here, we propose a model whereby S. aureus obtains sufficient metals to properly populate SODs and defend against superoxide during initial colonization. Following colonization and the arrival of neutrophils expressing CP, abscess development leads to Mn and Zn depletion, which in turn reduces bacterial defenses against superoxide and renders S. aureus more susceptible to neutrophil-mediated killing.

In addition to demonstrating that CP enhances the sensitivity of bacteria to superoxide, these results provide important insights into the relevance of the nutritional metal binding properties of CP. ITC analysis of Mn2+ and Zn2+ binding to WT CP and the ΔZn/Mn mutant shows that CP possesses two non-identical sites for binding these metals. The tight (nM) binding of CP for Mn2+ suggests that it effectively competes with bacterial Mn transport systems, which also possess affinities in the nM range (Papp-Wallace and Maguire, 2006). Interestingly, the anti-fungal activity of CP requires the C-terminal histidine-rich tail of S100A9, which was interpreted to implicate this region as a Zn2+ binding site (Sohnle et al., 2000). However, this supposition is not supported by ITC analysis, which showed no appreciable binding of Zn2+ or Mn2+ by ΔZn/Mn despite the presence of the histidine-rich tail.

Mn- or Cu-Zn-dependent SODs are found in a wide range of bacterial pathogens including Streptococcus pneumoniae, Salmonella typhimurium, Yersinia enterocolitica, and Neisseria meningitidis, suggesting that CP may enhance the superoxide sensitivity of a large number of medically relevant organisms (Fang et al., 1999; Lynch and Kuramitsu, 2000; Roggenkamp et al., 1997; Yesilkaya et al., 2000). This proposal is supported by the observation that Escherichia coli strains unable to acquire Mn display reduced Mn-dependent SOD activity despite Mn-SOD expression (Anjem et al., 2009). The ability of the host to limit metal availability and inhibit bacterial SODs may also provide an explanation for why some bacterial pathogens express multiple SODs with different metal dependencies (Lynch and Kuramitsu, 2000). As superoxide scavenging does not completely reverse the antimicrobial effects of CP other bacterial Mn- and Zn-dependent processes are likely disrupted as well (Andreini et al., 2006; Papp-Wallace and Maguire, 2006).

Our results show that CP reduces the activity of bacterial processes and proteins that require Mn. Further characterization of the bacterial processes disrupted by CP will enhance our understanding of how Mn and Zn sequestration by the host limits pathogenesis. Developing a greater understanding of the effect that metal sequestration has on invading pathogens will lay the groundwork for the development of therapeutics that target bacterial nutrient acquisition.

Experimental Procedures

Calprotectin activity assays

To determine the 50% inhibitory concentration of CP, S. aureus cultures were back diluted 1/50 from an overnight culture into 5 ml fresh TSB and grown for 1 hr. The bacteria were then back diluted 1/100 in 38% TSB, 62% CP buffer (CPB) (100 mM NaCl, 3 mM CaCl2 5 mM β-mecaptoethanol, 20 mM Tris pH 7.5) and grown for 7 hrs in 96 well plates. Absorbance at OD600 was measured to evaluate growth.

Superoxide stress assays

For paraquat assays, overnight cultures were diluted 1/100 into growth media containing 38% brain heart infusion broth (BHI) + 0.5% glucose and 62% CPB supplemented with CP, Mn, Zn, or glutathione as indicated and grown as for the IC50 assays. For xanthine/xanthine oxidase assays, bacteria were grown as for the paraquat assays with the addition 10 µM MnCl2. At stationary phase, the bacteria were harvested and washed twice with PBS, then resuspended to ~1×108 cfu/ml. Xanthine and xanthine oxidase (Sigma-Aldrich St. Louis, MO) were prepared as 5X and 10X stocks in PBS respectively. 50 µl of bacteria, 2 mM xanthine (final concentration) and 1.5 or 0.15 units of xanthine oxidase were combined in 100 µl total volume of PBS. These reactions were then incubated for 1 hr at 37 °C. The percent viable bacteria was determined by plating serial dilutions and comparing to the inoculums.

Cellular SOD and superoxide assays

For SOD activity assays, bacteria were grown as for the paraquat assays. Samples were harvested at appropriate time points and washed and resuspended in 25 mM Tris pH 8.0. Bacteria were then lysed via mechanical disruption and cellular debris was removed by centrifugation as previously described (Valderas and Hart, 2001). Superoxide dismutase activity and protein in the supernatant was determined using the SOD Assay Kit-WST (Sigma-Aldrich St. Louis, MO), BCA assay (Peirce Thermo-Fisher Rockford, IL) respectively. Mn-dependent SOD purified from E. coli (Sigma-Aldrich St. Louis, MO) was used as a standard.

Intracellular superoxide levels were determined using the membrane permeable dye dihydroethidium. S. aureus was grown as above in the presence and absence of CP and then incubated with 1/10 volume 0.1 M DHE dissolved in DMSO or DMSO alone for 35 min. The bacteria were then washed and resuspended in PBS and analyzed via FACS using excitation wavelengths of 405 nm and 488 nm and emission wavelengths of 440 nm and 695 nm to measure uptake of DHE and formation of ethidium bromide, respectively.

Infections and Neutrophil killing assays

All animal experiments were approved by the Vanderbilt Medical Center Institutional Animal Care and Use Committee. Mouse infections were performed as previously described, details are provided in the supplemental (Corbin et al., 2008). For neutrophil killing assays peritoneal PMNs were elicited using casein and harvested from 13–16 week old male C57BL/6 mice (Jackson Labs) and bacteria were prepared essentially as previously described (Corbin et al., 2008; Luo and Dorf, 1997). Bacteria were grown as for the superoxide assays to either exponential or stationary phase with the addition of 10 µM MnCl2 and calprotectin as appropriate, details are provided in the supplemental.

Statistical Analyses

Statistical significance was assessed by using either the Student’s t-test (Graphpad Prism V5 or Excel 2007) or 1 way and 2 way ANOVA (Graphpad V5) as indicated. Results were considered statistically significant if the P-value was less than 0.05.

Highlights.

  • Calprotectin (CP) increases S. aureus sensitivity to superoxide stress

  • Mn/Zn binding is required for the antimicrobial activity of CP

  • Staphylococcal Mn-dependent superoxide defenses are inhibited by CP

  • CP inhibition of superoxide defenses enhances S. aureus killing by neutrophils

Supplementary Material

01

Acknowledgments

This publication was made possible by grants from the US NIH: U54 AI057157 from the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense R01AI069233 and R01AI073843 to EPS; 1 R01GM62112 (S1) to WJC; an NIH (T32 HL094296-02) American Heart Association postdoctoral fellowship to TKF; a Canadian Institutes of Health Research fellowship to SC; Public Health Service award T32 GM07347 MIH; a NIH postdoctoral fellowship (5 T32 CA009582-23) to SD; and CG Vanderbilt NSF-REU program (CHE 0850976). The manuscript’s contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. We thank Dr. Mark Hart for providing us with the sod alleles and Dr. Jonathan Sheehan for helpful discussions.

Footnotes

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The authors have no conflicting financial interests.

References

  1. Andreini C, Banci L, Bertini I, Rosato A. Zinc through the three domains of life. J Proteome Res. 2006;5:3173–3178. doi: 10.1021/pr0603699. [DOI] [PubMed] [Google Scholar]
  2. Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM. Metal ions in biological catalysis: from enzyme databases to general principles. J Biol Inorg Chem. 2008;13:1205–1218. doi: 10.1007/s00775-008-0404-5. [DOI] [PubMed] [Google Scholar]
  3. Anjem A, Varghese S, Imlay JA. Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli. Molecular microbiology. 2009;72:844–858. doi: 10.1111/j.1365-2958.2009.06699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Babior BM. NADPH oxidase. Curr Opin Immunol. 2004;16:42–47. doi: 10.1016/j.coi.2003.12.001. [DOI] [PubMed] [Google Scholar]
  5. Carter WO, Narayanan PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol. 1994;55:253–258. doi: 10.1002/jlb.55.2.253. [DOI] [PubMed] [Google Scholar]
  6. Clements MO, Watson SP, Foster SJ. Characterization of the major superoxide dismutase of Staphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity. J Bacteriol. 1999;181:3898–3903. doi: 10.1128/jb.181.13.3898-3903.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clohessy PA, Golden BE. Calprotectin-mediated zinc chelation as a biostatic mechanism in host defence. Scand J Immunol. 1995;42:551–556. doi: 10.1111/j.1365-3083.1995.tb03695.x. [DOI] [PubMed] [Google Scholar]
  8. Corbin BD, Seeley EH, Raab A, Feldmann J, Miller MR, Torres VJ, Anderson KL, Dattilo BM, Dunman PM, Gerads R, et al. Metal chelation and inhibition of bacterial growth in tissue abscesses. Science. 2008;319:962–965. doi: 10.1126/science.1152449. [DOI] [PubMed] [Google Scholar]
  9. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, Lin F, Lin J, Carleton HA, Mongodin EF, et al. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet. 2006;367:731–739. doi: 10.1016/S0140-6736(06)68231-7. [DOI] [PubMed] [Google Scholar]
  10. Fang FC, DeGroote MA, Foster JW, Baumler AJ, Ochsner U, Testerman T, Bearson S, Giard JC, Xu Y, Campbell G, et al. Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases. Proc Natl Acad Sci U S A. 1999;96:7502–7507. doi: 10.1073/pnas.96.13.7502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gebhardt C, Nemeth J, Angel P, Hess J. S100A8 and S100A9 in inflammation and cancer. Biochem Pharmacol. 2006;72:1622–1631. doi: 10.1016/j.bcp.2006.05.017. [DOI] [PubMed] [Google Scholar]
  12. Grundmann H, Aires-de-Sousa M, Boyce J, Tiemersma E. Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet. 2006;368:874–885. doi: 10.1016/S0140-6736(06)68853-3. [DOI] [PubMed] [Google Scholar]
  13. Horsburgh MJ, Wharton SJ, Cox AG, Ingham E, Peacock S, Foster SJ. MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake. Mol Microbiol. 2002a;44:1269–1286. doi: 10.1046/j.1365-2958.2002.02944.x. [DOI] [PubMed] [Google Scholar]
  14. Horsburgh MJ, Wharton SJ, Karavolos M, Foster SJ. Manganese: elemental defence for a life with oxygen. Trends Microbiol. 2002b;10:496–501. doi: 10.1016/s0966-842x(02)02462-9. [DOI] [PubMed] [Google Scholar]
  15. Karavolos MH, Horsburgh MJ, Ingham E, Foster SJ. Role and regulation of the superoxide dismutases of Staphylococcus aureus. Microbiology. 2003;149:2749–2758. doi: 10.1099/mic.0.26353-0. [DOI] [PubMed] [Google Scholar]
  16. Kehl-Fie TE, Skaar EP. Nutritional immunity beyond iron: a role for manganese and zinc. Current opinion in chemical biology. 2010;14:218–224. doi: 10.1016/j.cbpa.2009.11.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998;339:520–532. doi: 10.1056/NEJM199808203390806. [DOI] [PubMed] [Google Scholar]
  18. Luo Y, Dorf ME. Isolation of Mouse Neutrophils. Curr protoc Immuno. 1997;22 doi: 10.1002/0471142735.im0320s22. 3.20.21-23.20.26. [DOI] [PubMed] [Google Scholar]
  19. Lynch M, Kuramitsu H. Expression and role of superoxide dismutases (SOD) in pathogenic bacteria. Microbes Infect. 2000;2:1245–1255. doi: 10.1016/s1286-4579(00)01278-8. [DOI] [PubMed] [Google Scholar]
  20. Papp-Wallace KM, Maguire ME. Manganese transport and the role of manganese in virulence. Annu Rev Microbiol. 2006;60:187–209. doi: 10.1146/annurev.micro.60.080805.142149. [DOI] [PubMed] [Google Scholar]
  21. Roggenkamp A, Bittner T, Leitritz L, Sing A, Heesemann J. Contribution of the Mn-cofactored superoxide dismutase (SodA) to the virulence of Yersinia enterocolitica serotype O8. Infect Immun. 1997;65:4705–4710. doi: 10.1128/iai.65.11.4705-4710.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Said-Salim B, Mathema B, Kreiswirth BN. Community-acquired methicillin-resistant Staphylococcus aureus: an emerging pathogen. Infect Control Hosp Epidemiol. 2003;24:451–455. doi: 10.1086/502231. [DOI] [PubMed] [Google Scholar]
  23. Sohnle PG, Hunter MJ, Hahn B, Chazin WJ. Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14) J Infect Dis. 2000;182:1272–1275. doi: 10.1086/315810. [DOI] [PubMed] [Google Scholar]
  24. Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, Brinkmann V, Jungblut PR, Zychlinsky A. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS pathogens. 2009;5:e1000639. doi: 10.1371/journal.ppat.1000639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Valderas MW, Gatson JW, Wreyford N, Hart ME. The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci. J Bacteriol. 2002;184:2465–2472. doi: 10.1128/JB.184.9.2465-2472.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Valderas MW, Hart ME. Identification and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus. J Bacteriol. 2001;183:3399–3407. doi: 10.1128/JB.183.11.3399-3407.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weinberg ED. Iron availability and infection. Biochim Biophys Acta. 2009;1790:600–605. doi: 10.1016/j.bbagen.2008.07.002. [DOI] [PubMed] [Google Scholar]
  28. Yesilkaya H, Kadioglu A, Gingles N, Alexander JE, Mitchell TJ, Andrew PW. Role of manganese-containing superoxide dismutase in oxidative stress and virulence of Streptococcus pneumoniae. Infect Immun. 2000;68:2819–2826. doi: 10.1128/iai.68.5.2819-2826.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yousefi R, Imani M, Ardestani SK, Saboury AA, Gheibi N, Ranjbar B. Human calprotectin: effect of calcium and zinc on its secondary and tertiary structures, and role of pH in its thermal stability. Acta Biochim Biophys Sin (Shanghai) 2007;39:795–802. doi: 10.1111/j.1745-7270.2007.00343.x. [DOI] [PubMed] [Google Scholar]

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