A Superoxide Dismutase Capable of Functioning with Iron or Manganese Promotes the Resistance of Staphylococcus aureus to Calprotectin and Nutritional Immunity

Abstract

Staphylococcus aureus is a devastating mammalian pathogen for which the development of new therapeutic approaches is urgently needed due to the prevalence of antibiotic resistance. During infection pathogens must overcome the dual threats of host-imposed manganese starvation, termed nutritional immunity, and the oxidative burst of immune cells. These defenses function synergistically, as host-imposed manganese starvation reduces activity of the manganese-dependent enzyme superoxide dismutase (SOD). S. aureus expresses two SODs, denoted SodA and SodM. While all staphylococci possess SodA, SodM is unique to S. aureus, but the advantage that S. aureus gains by expressing two apparently manganese-dependent SODs is unknown. Surprisingly, loss of both SODs renders S. aureus more sensitive to host-imposed manganese starvation, suggesting a role for these proteins in overcoming nutritional immunity. In this study, we have elucidated the respective contributions of SodA and SodM to resisting oxidative stress and nutritional immunity. These analyses revealed that SodA is important for resisting oxidative stress and for disease development when manganese is abundant, while SodM is important under manganese-deplete conditions. In vitro analysis demonstrated that SodA is strictly manganese-dependent whereas SodM is in fact cambialistic, possessing equal enzymatic activity when loaded with manganese or iron. Cumulatively, these studies provide a mechanistic rationale for the acquisition of a second superoxide dismutase by S. aureus and demonstrate an important contribution of cambialistic SODs to bacterial pathogenesis. Furthermore, they also suggest a new mechanism for resisting manganese starvation, namely populating manganese-utilizing enzymes with iron.

Fig 1. Expression of sodA and sodM varies by Mn availability.

S. aureus carrying (A, C, & E) pSodA and (B, D, & F) pSodM YFP reporter constructs were grown in the presence of either wild type CP (A-F), the ΔMn/Zn site mutant (C-F), or the ΔZn site mutant (C-F), and in the (A, B, E, & F) presence or (A-D) absence of 0.1 mM PQ. Expression data are normalized to growth. Error bars indicate SEM (n = 3 or more). (A & B) # = p <0.05 via two-way ANOVA with Tukey’s post-test for the indicated comparison. * = p <0.05 via two-way ANOVA with Tukey’s post-test relative to bacteria grown in the presence of paraquat without CP. ^ = p <0.05 via two-way ANOVA with Tukey’s post-test relative to bacteria grown in the absence of both paraquat and CP (C-F) * = p <0.05 via two-way ANOVA with Dunnett’s post-test when compared to wild type CP.

Fig 2. SodM is the predominant source of SOD activity in Mn-deplete environments.

Wild type S. aureus was grown in various concentrations of CP in the (A & B) absence and (B) presence of 0.1 mM PQ, and both (A) the individual activities of the SODs and (B) the fractional contribution of SodA and SodM to total SOD activity in cell lysates (5.17 μg of total protein) was determined. The lower gel was treated with hydrogen peroxide prior to assessing SOD activity to inactivate Fe-containing SODs. Purified recombinant SodA and SodM (0.3 μg), loaded with either Mn or Fe, were included as controls. The experiment was repeated 3 times and representative gels are shown.

Fig 3. SodM is required to resist CP-imposed Mn starvation.

Wild type, ΔsodA, ΔsodM and ΔsodAΔsodM S. aureus were grown in the presence of increasing concentrations of (A & B) WT CP, (C) the ΔMn/Zn site mutant, or (D) the ΔZn site mutant in the (A) absence and (C-D) presence of 0.1 mM PQ. Growth was assessed by measuring optical density (OD₆₀₀). * = p<0.05 relative to wild type at the same concentration of CP via two-way ANOVA with Dunnett’s post-test. Error bars indicate SEM (n = 3 or more).

Fig 4. SodM can be metallated with either Mn or Fe in S. aureus.

The S. aureus ΔsodAΔsodM mutant expressing SodM from a plasmid was grown in NRPMI supplemented with either 1 μM FeCl₂ or 1 μM MnCl₂ and SOD activity was assessed in cell lysates (24.8 μg of total protein), with and without peroxide treatment. Peroxide treatment was used to inactivate Fe-containing SODs. Purified SodM (0.3 μg), loaded with either Mn or Fe, were included as controls. The experiment was repeated 3 times, and representative gels are shown.

Fig 5. SodM contributes to resisting Mn starvation during infection.

Wild type C57BL/6 (C57) and C57BL/6 S100A9^-/- (CP^-/-) mice were infected with either wild type, ΔsodA or ΔsodM S. aureus. Mice were sacrificed 96 h after infection, and bacterial loads in the livers were enumerated. p <0.05 as determined by Mann-Whitney test.