Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection

Abstract

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.

Figure 1. Postulated Siderophore Biosynthetic Pathway of A. fumigatus

Steps identified during this study are in blue.

Figure 2. Northern Analysis of A. fumigatus sidC, sidD, sidF, and sidG

Following growth for 24 h during iron starvation (−Fe) and sufficient iron (+Fe), total RNA was isolated from A. fumigatus ATCC46645. As a loading control, blots were hybridized with the β-tubulin encoding tubA gene of A. fumigatus.

Figure 3. Extra- and Intracellular Siderophore Production of A. fumigatus wt, ΔsidA, ΔsidC, ΔsidD, ΔsidF, and ΔsidG

(A) Representative HPLC analysis of culture supernatants, cell extracts, and conidial extracts of the wt. Units are given in milli absorption units (mAu). (B) Quantification of siderophore production of ΔsidA, ΔsidC, ΔsidD, ΔsidF, and ΔsidG normalized to that of the wt after 24 h of growth in iron-depleted conditions. For HPLC analysis of the conidial siderophore, the wt and mutant strains were grown for 5 d either with 0.5 mM FeSO₄, 10 μM FC (^FC), or 10 μM TAFC (^TAFC), respectively, as iron source. ¹The FSC and FC contents in wt supernatant represented 12.0±3.1% and 1.5±3.1% of the TAFC content, respectively. The data represent the means ± standard deviations of results from three independent experiments. ND, not detected; na, not analyzed.

Figure 4. Time Course Analysis of Siderophore Content and of brlA Expression during Conidiation

A. fumigatus wt cells were grown for 24 h under iron-replete liquid culture and subsequently transferred to iron-replete solid media. After growth on solid media up to the indicated time points, FC and HFC content was analyzed, as well as brlA expression. As a loading control, tubA was used. Quantity units of siderophore determination are given in milli absorption units (mAu).

Figure 5. A. fumigatus Conidiation Rates in wt and Siderophore Biosynthetic Mutant Backgrounds

10⁶ conidia of fungal strains were point inoculated in the center of minimal medium plates containing the indicated iron source. Conidia produced by 1 cm² were counted after 120 h of incubation at 37 °C. The wt conidia count was 4.5 × 10⁸. The data represent the means ± standard deviations of results from three independent experiments.

Figure 6. Impact of Extra- and Intracellular Siderophores on Resistance to Iron Limitation and Oxidative Stress

(A) 10⁴ conidia of wt were point inoculated and radial growth was measured after 48 h at 37 °C on minimal medium lacking iron (−Fe), containing 10 μM FeSO₄ (+Fe), 0.5 mM FeSO₄ (hFe), 0.25 mM bathophenanthroline disulfonate (BPS), 2 mM H₂O₂, respectively. (B) Radial growth of respective mutant strains was determined as described in (A) and normalized to that of the wt grown in the same condition. The data in (A, B) represent the means ± standard deviations of results from three independent experiments. (C, D) Analysis of hydrogen peroxide sensitivity of conidia (C) and hyphae (D) was determined as described in Materials and Methods. The conidia used were harvested from plates containing 1.5 mM FeSO₄ or 10 μM FC (^FC). Samples were prepared in triplicate, and the standard deviation did not exceed 15%.

Figure 7. CatA Activity in wt, ΔsidA, ΔsidC, ΔsidD, ΔsidF, and ΔsidG

A 40-μg conidial protein extract prepared from spores harvested from sporulation medium containing either 1.5 mM FeSO₄ or 10 μM FC as iron source, respectively, was subject to native polyacrylamide gel electrophoresis (PAGE) and catalase (ferricyanide-negative) staining as described by Paris et al. [29]. As a control for loading and protein quality, the same samples were alternatively subject to SDS-PAGE (8%) and Coomassie staining (CS). Lane A, ΔsidA; lane C, ΔsidC; lane D, ΔsidD; lane F, ΔsidF; lane G, ΔsidG. M, molecular mass marker lane.

Figure 8. Analysis of Murine Survival following A. fumigatus Siderophore Mutant Infection

Comparative survival of neutropenic mice following infection with A. fumigatus ΔsidC, ΔsidD, ΔsidF, and ΔsidG (broken lines) and corresponding complemented strains (solid lines). Mice were sacrificed when 20% of body weight with respect to the day of infection was reached.

Figure 9. Histopathological Analysis of Aspergillus-Infected Murine Lung Sections

Comparative histopathology of neutropenic murine lung sections following infection with A. fumigatus wt or A. fumigatus ΔsidA, ΔsidC, ΔsidD, ΔsidF, and ΔsidG mutants. Sections were sampled at 24 and 72 h post-infection, fixed in 4% ^v/_v formaldehyde, and stained using Grocotts Methanamine Silver (GMS), or hematoxylin and eosin (HE). Infectious foci containing fungal hyphae and inflammatory lesions are indicated by arrows over GMS and HE sections, respectively.

Figure 10. HFC-Mediated Rescue of ΔsidA Virulence in Neutropenic Mice

Comparative survival of neutropenic mice (left panel) following infection with FC-supplemented (ΔsidA^FC) and non-supplemented (ΔsidA) ΔsidA conidia, representing FC-loaded and unloaded conidia, respectively. Histopathological analysis (right panel) of Grocotts Methanamine Silver–stained tissue sections at 4 d post-infection reveals discreet mycelial lesions.