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

Abstract

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.

Figure 1. Calprotectin enhances the effects of superoxide stress

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)

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

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)

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

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)

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

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.