Figure 3. Zn and Mn binding contribute to calprotectin Anti Aspergillus activity.

A. fumigatus hyphae were incubated +/− recombinant calprotectin for 16h and fungal growth was assessed by calcofluor staining (A-D). % Fungal mass was calculated by fluorimetry of calcofluor and shown as percent of total hyphae when grown in media alone (experimental/control × 100). A. Representative images of hyphae (200X) in media +/− 25 µg/ml calprotectin B. Quantification of hyphael growth + 50 µg/ml calprotectin +/− increasing ZnSO₄ +/− 1 or 5 µM MnSO₄C-D. Quantification of hyphal growth of hyphae incubated with wild-type calprotectin (CP) vs. CPΔMn calprotectin (lacks Mn binding motif; note difference in the y axis scale) (C) or CP vs. or CPΔMn/Zn calprotectin (lacks Mn and Zn binding motifs). E-FA. fumigatus hyphae were incubated with 2 × 10⁵ human neutrophils in RPMI +/− 5 µM ZnSO₄ or MnSO₄ and fungal growth was assessed by calcofluor staining. E. % Fungal mass was calculated by fluorimetry of calcofluor and shown as percent of total hyphae when grown in media alone (experimental/control × 100). F. Representative images of hyphae (200X) in media +/− neutrophils, ZnSO₄ or MnSO_4. Representative In vitro experiments with 3–6 technical replicates per condition, significance was determined by student’s t-test. Human neutrophils were tested from three separate donors.