AFU cannot retrieve iron from holo-transferrin, and enhanced labile plasma iron uptake depends on fungal siderophores. (A) Role of holo-Tf as an iron source for AFU growth. Representative microscopy images of AFU cultures taken 48 hours after inoculation (5 × 10⁴ spores/mL) of RPMI containing 10% ferric iron-spiked human plasma (0-50 µM Fe³⁺) supplemented with 2,5 mg/mL apo-Tf or holo-Tf, respectively (original magnification ×20; bars represent 30 µm). (B-C) Removal of iron from holo-Tf by AFU was studied. AFU spores (5 × 10⁴/mL) were inoculated into RPMI containing 5µM Fe³⁺ and 2 mg/mL apo- or holo-Tf in 6-well plates. Holo-Tf:apo-Tf conversion in culture supernatant was monitored by absorbance measurements at 280 nm (A_280corr) and 460 nm (A_460corr) (B) and urea–polyacrylamide gel electrophoresis (C) at indicated times. Uninoculated RPMI media with apo- and holo-Tf were used as controls in both assays. (D) eLPI use by AFU mutant strains with defects in iron acquisition systems. Representative microscopy images showing the growth pattern of wild-type (Wt), ΔsidF, and ΔsidA conidia in RPMI containing 10% ferric iron-spiked human plasma after 48-hour culture (original magnification ×20; bars represent 30 µm). (E) Influence of clinically applicable iron chelators on AFU outgrowth in eLPI-deficient and eLPI-positive serum cultures. AFU spores (5 × 10⁴/mL) were cultured in RPMI containing 10% ferric iron-spiked human plasma supplemented with 100 µM deferoxamine (DFO) or 200 µM deferasirox (DFX). Control cultures were DMSO treated. Photographs were taken 48 hours after inoculation. Representative images of fungal cultures are shown (original magnification ×20; bars represent 30 µm). Experiments shown in panels A, D, and E were performed at least in 6 replicates, all showing consistent results.