Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens Original paper

What was studied?

This original research article investigated how the neutrophil protein complex calprotectin (CP), a heterodimer of S100A8/S100A9, enforces “nutritional immunity” by withholding essential metals from bacteria, with a focus on defining the molecular mechanism of manganese sequestration by calprotectin. Using a combined biophysical–structural–microbiological approach, the authors engineered site-specific CP mutants to disentangle zinc (Zn²⁺) versus manganese (Mn²⁺) binding, quantified metal affinities by isothermal titration calorimetry, and tested antimicrobial activity in bacterial growth assays. A high-resolution (1.6 Å) X-ray crystal structure of Mn-bound CP revealed an unusual Mn²⁺-binding architecture that explains why CP, unlike other S100 proteins, can chelate Mn²⁺ with very high affinity. Collectively, the work clarifies how manganese sequestration by calprotectin functions as a core component of innate immune defense against diverse pathogens.

Who was studied?

No human participants were enrolled; instead, the study examined recombinant human calprotectin proteins and multiple clinically relevant bacterial pathogens. The primary mechanistic bacterial model was Staphylococcus aureus, including experiments initiated from both exponential- and stationary-phase cultures to mimic different physiological states relevant to infection. To test whether findings generalized beyond a single organism, the authors evaluated a spectrum of Gram-positive and Gram-negative pathogens: Staphylococcus epidermidis, Staphylococcus lugdunensis, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Shigella flexneri. This design allowed the team to assess whether manganese sequestration by calprotectin is broadly required for growth inhibition across bacterial taxa, not merely in S. aureus.

Most important findings

Calprotectin contains two high-affinity Zn²⁺-binding sites but only one high-affinity Mn²⁺ site, and the Mn²⁺ site is specifically the noncanonical “Site 1” (S1). Mutational knockout of S1 (ΔS1) preserved only Zn²⁺ chelation and abolished Mn²⁺ binding, whereas knockout of the canonical “Site 2” (ΔS2) preserved Mn²⁺ binding. Functionally, Zn²⁺ sequestration alone was not sufficient for maximal bacterial growth inhibition: ΔS1 could not fully suppress S. aureus growth even at high concentrations, while ΔS2 inhibited similarly to wild-type CP, demonstrating that Mn²⁺ deprivation is necessary for full antimicrobial activity. Mechanistically, Mn²⁺ sequestration directly impaired S. aureus oxidative stress defenses: wild-type CP and ΔS2 (Mn-binding competent) increased sensitivity to paraquat, raised intracellular superoxide, and reduced superoxide dismutase activity—effects not seen with ΔS1 and reversible with Mn²⁺ addback. Structurally, the Mn²-bound CP crystal structure showed a unique six-histidine, near-perfect octahedral coordination sphere formed by residues from both subunits plus two histidines from the distinctive S100A9 C-terminal tail, a feature not previously observed for Mn²⁺ in the Protein Data Bank. Tail truncation (ΔTail) eliminated Mn²⁺ binding while preserving Zn²⁺ binding and similarly reduced antimicrobial potency, reinforcing that the tail-enabled Mn²⁺ site underpins broad-spectrum activity. Across multiple pathogens, the Mn-binding competent variant inhibited growth comparably to wild-type CP, whereas the Mn-binding deficient mutant showed consistently weaker inhibition, supporting Mn²⁺ restriction as a key and generalizable antimicrobial mechanism.

Key implications

For clinicians and microbiome-focused interpretation, this paper provides a mechanistic bridge between a widely used inflammatory biomarker and a concrete antimicrobial function: manganese sequestration by calprotectin is not merely a correlate of inflammation but a causal antibacterial strategy. Because fecal and mucosal calprotectin are routinely used to gauge intestinal inflammation, these findings support viewing elevated calprotectin as a marker of an environment with intensified metal restriction pressure—especially Mn²⁺—that can reshape microbial community structure by favoring organisms with superior Mn²⁺ uptake systems or alternative oxidative stress strategies. The work also identifies Mn²-dependent bacterial defenses (notably superoxide dismutase activity) as therapeutic vulnerabilities, implying that interventions amplifying Mn²⁺ restriction or targeting Mn²⁺ acquisition pathways may synergize with innate immunity and potentially reduce reliance on conventional antibiotics.

Citation

Damo SM, Kehl-Fie TE, Sugitan N, et al. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens.Proc Natl Acad Sci U S A. 2013;110(10):3841-3846. doi:10.1073/pnas.1220341110