Zinc and manganese chelation by neutrophil S100A8/A9 (calprotectin) limits extracellular Aspergillus fumigatus hyphal growth and corneal infection Original paper

What was studied?

This original research article tested whether calprotectin limits Aspergillus hyphal growth during infection, and which metal-binding functions explain that activity. Using a murine corneal infection (fungal keratitis) model plus in vitro hyphal growth assays, the authors examined neutrophil-derived calprotectin (the S100A8/S100A9 heterodimer) as a mechanism of “nutritional immunity,” specifically zinc (Zn) and manganese (Mn) sequestration. They also used engineered calprotectin mutants that selectively lose Zn/Mn binding, and Aspergillus fumigatus strains with impaired Zn uptake (ΔzafA) to test whether fungal metal-acquisition pathways determine susceptibility to host chelation.

Who was studied?

The work was preclinical and mechanistic. In vivo experiments used C57BL/6 wild-type mice and calprotectin-deficient S100A9 knockout mice (S100A9⁻/⁻), infected intrastromally with dsRed-expressing A. fumigatus conidia to quantify fungal burden and hyphal mass in corneas at 24–48 hours post-infection. Additional mouse models included CD18-deficient mice (impaired neutrophil transmigration) and bone marrow chimeras to assess pulmonary challenge outcomes. In vitro assays used purified mouse peritoneal neutrophils and neutrophil lysates, as well as neutrophils from healthy adult human donors (18–65 years) to confirm conserved antifungal effects and metal-dependence.

Most important findings

Calprotectin was essential for controlling extracellular hyphal growth in the cornea but was dispensable for intracellular conidial uptake and killing. S100A9⁻/⁻ mice developed significantly higher fungal burden and CFU in infected corneas despite similar neutrophil recruitment, implying a qualitative defect in antifungal function rather than impaired inflammation. Recombinant calprotectin delivered locally restored protection, directly reducing hyphal mass in S100A9⁻/⁻ corneas. Mechanistically, antihyphal activity required metal chelation: wild-type calprotectin strongly inhibited hyphae; a mutant lacking both Zn and Mn binding (CPΔZn/Mn) lost antifungal activity, while a Mn-binding–defective mutant (CPΔMn) showed only partial loss—supporting Zn chelation as primary with Mn as contributory. Hyphal growth could be rescued by adding Zn (fully) and only partially by Mn alone, and human neutrophil inhibition was reversed by Zn but not Mn. Finally, the fungal ΔzafA mutant (defective Zn transport regulation) was hypersusceptible to calprotectin/neutrophils and showed reduced virulence in the cornea, linking a specific microbial nutrient-acquisition program to a host-derived metal-sequestration pressure.

Key implications

Clinically, this study highlights a stage-specific antifungal defense: calprotectin-driven Zn/Mn sequestration targets extracellular hyphae—precisely the invasive, tissue-damaging form relevant to fungal keratitis—while not materially influencing early conidial handling in an immunocompetent lung model. For a microbiome/signatures database, the key actionable association is a host factor (calprotectin) that suppresses A. fumigatus growth by depriving essential metals, plus a microbial counterstrategy (ZafA-regulated Zn uptake) that predicts resistance and virulence. These findings support therapeutic concepts that augment local metal sequestration (e.g., topical calprotectin-like strategies) or inhibit fungal Zn acquisition pathways as adjuncts to antifungal drugs, especially in settings where hyphae dominate (corneal infection, neutropenia, or impaired neutrophil function).

Citation

Clark HL, Jhingran A, Sun Y, et al. Zinc and manganese chelation by neutrophil S100A8/A9 (calprotectin) limits extracellular Aspergillus fumigatus hyphal growth and corneal infection.J Immunol. 2016;196:000-000. Published online November 18, 2015. doi:10.4049/jimmunol.1502037