Iron-regulated siderophore production and expression of A. fumigatus sidA, ftrA, and fetC. (A) Siderophore biosynthesis is up-regulated during iron starvation. TAFC, DFFC, and FC levels in both wild-type A. fumigatus strains CEA10 (1) and ATCC 46645 (2), following growth for 24 h at 37°C in AMM (20) in iron starvation (−Fe) and iron-replete (+Fe; 10 μM FeSO₄) conditions, were determined by reversed phase HPLC analysis as described previously (21). A. fumigatus CEA10 siderophore levels under iron starvation conditions were normalized to 100%. (B) A. fumigatus sidA, ftrA, and fetC expression is up-regulated during iron starvation. Total RNA was isolated from A. fumigatus CEA10 following growth for 24 h during iron starvation (−Fe) and iron-replete (+Fe) conditions and subjected to Northern analysis. As a control for loading and RNA quality, blots were hybridized with a fragment from the β-tubulin encoding tubA gene. (C) A. fumigatus sidA–deficient strains do not synthesize siderophores, and ftrA deficiency brings forward the onset of siderophore biosynthesis. The siderophore production of A. fumigatus wild-type (CEA10 [1], ATCC 46645 [6]) and mutant strains (ΔsidA-CEA17 [2], sidA^c-CEA17 [3], ΔftrA-CEA17 [4], ΔsidA/ΔftrA-CEA17 [5], ΔsidA [7], sidA^R [8], ΔftrA [9]) was analyzed following growth for 12 or 24 h under iron starvation conditions. A. fumigatus CEA10 siderophore levels at 24 h were normalized to 100%.

Virulence analysis of A. fumigatus iron uptake mutants. Siderophore biosynthesis is essential, whereas reductive iron uptake is unnecessary for A. fumigatus virulence. Groups of 15 neutropenic CD1 mice were infected with 2 × 10⁵ conidiospores. ΔftrA (closed triangles) and sidA^R (closed circles) demonstrate comparable virulence (P = 0.1656 and P = 0.9617, respectively, by Kaplan Meier log rank analysis) to the wild-type strain ATCC 46645 (open squares). In comparison to A. fumigatus ATCC 46645 and sidA^R, ΔsidA (closed circles) is completely attenuated for virulence (P < 0.0001).

(A) Growth phenotypes of A. fumigatus wild-type and mutant strains. SidA and FtrA are components of two independent iron uptake mechanisms. Aliquots of 10⁴ conidia of the respective strain were point inoculated on AMM plates containing the respective iron source and incubated for 48 h at 37°C. Blood agar was AMM containing 5% (vol/vol) sheep blood. (B) Quantitative analysis of radial growth of A. fumigatus wild-type (CEA10 [1], ATCC 46645 [6]) and mutant strains (ΔsidA-CEA17 [2], sidA^c-CEA17 [3], ΔftrA-CEA17 [4], ΔsidA/ΔftrA-CEA17 [5], ΔsidA [7], sidA^R [8], ΔftrA [9]) was performed following growth for 48 h at 37°C. A. fumigatus CEA10 radial growth on 10 μM FeSO₄ was normalized to 100%. (C) Quantitative analysis of conidiation of A. fumigatus wild-type (CEA10 [1], ATCC 46645 [6]) and mutant strains (ΔsidA-CEA17 [2], sidA^c-CEA17 [3], ΔftrA-CEA17 [4], ΔsidA/ΔftrA-CEA17 [5], ΔsidA [7], sidA^R [8], ΔftrA [9]) was performed following growth for 120 h at 37°C. A. fumigatus CEA10 conidiation rate on 10 μM FeSO₄ was normalized to 100%.

Histological analysis of mice infected with A. fumigatus ΔsidA and sidA^R strains. The A. fumigatus ΔsidA mutant does not germinate in vivo and causes minimal cellular infiltration, whereas the sidA^R strain germinates, grows profusely, engenders a massive cellular infiltration, and causes major tissue damage. Lungs were surgically removed from mice killed 60 h postinfection with 2 × 10⁵ A. fumigatus ΔsidA (A and B) or sidA^R (C and D). Sequential paraffin-embedded murine lung sections were stained with light green and Grocott's methenamine silver (A and C) or hematoxylin and eosin (B and D).