Targeting Iron Acquisition Blocks Infection with the Fungal Pathogens Aspergillus fumigatus and Fusarium oxysporum

Filamentous fungi are an important cause of pulmonary and systemic morbidity and mortality, and also cause corneal blindness and visual impairment worldwide. Utilizing in vitro neutrophil killing assays and a model of fungal infection of the cornea, we demonstrated that Dectin-1 dependent IL-6 production regulates expression of iron chelators, heme and siderophore binding proteins and hepcidin in infected mice. In addition, we show that human neutrophils synthesize lipocalin-1, which sequesters fungal siderophores, and that topical lipocalin-1 or lactoferrin restricts fungal growth in vivo. Conversely, we show that exogenous iron or the xenosiderophore deferroxamine enhances fungal growth in infected mice. By examining mutant Aspergillus and Fusarium strains, we found that fungal transcriptional responses to low iron levels and extracellular siderophores are essential for fungal growth during infection. Further, we showed that targeting fungal iron acquisition or siderophore biosynthesis by topical application of iron chelators or statins reduces fungal growth in the cornea by 60% and that dual therapy with the iron chelator deferiprone and statins further restricts fungal growth by 75%. Together, these studies identify specific host iron-chelating and fungal iron-acquisition mediators that regulate fungal growth, and demonstrate that therapeutic inhibition of fungal iron acquisition can be utilized to treat topical fungal infections

The effect of iron dextran, deferroxamine and lactoferrin on <i>A. fumigatus</i> corneal infection.

<p><b>A.</b> Serum iron levels 24 h after corneal infection. C57BL/6 mice were pre-treated at day -2, and day-1 with I.P. injections of iron-dextran (Fe-Dextran) or deferroxamine (Defox), and serum iron levels were measured by spectrophotometry (data are mean +/− SD of 5 mice per group). <b>B.</b> Fungal growth (dsRed <i>A. fumigatus</i>) and corneal opacity in mice given Fe-Dextran or Defox. <b>C.</b> Metamorph image analyses of dsRed fluorescence. <b>D.</b> Colony forming units (CFU) per eye at 4 h and 48 h post-infection <b>E,F.</b> Metamorph image analyses of percent (<b>E</b>) and total (<b>F</b>) corneal opacification. <b>G–I:</b> Effect of lactoferrin on fungal growth. C57BL/6 mice were infected with <i>A. fumigatus</i> dsRed conidia, and given topical lactoferrin (10.4 µg) at 0 and 6 h post-infection. Corneas were examined after 24 h. <b>G:</b> representative images; <b>H.</b> image analysis of dsRed expression, and <b>I.</b> CFU per eye. Panels B and G show representative images, and data points in panels C–F, H, I represent individual corneas. All panels show representative data from one experiment except for panels D and I which show pooled data from repeat experiments. These experiments were repeated three times with similar results.</p

Effect of topical simvastatin and deferiprone on fungal infection.

<p><b>A.</b> Statin targeting of fungal HMG-CoA reductase in siderophore biosynthesis. <b>B, C.</b> Effect of statins and iron chelators on growth of <i>A. fumigatus</i> and <i>F. oxysporum in vitro</i>. <b>B.</b> Simvastatin, lovastatin, deferiprone, or deferroxamine were added to growing cultures of <i>A. fumigatus</i> or <b>C. </b><i>F. oxysporum</i> for 16 h, and hyphal growth was quantified by calcofluor white. <b>D.</b> Growth of <i>A. fumigatus</i> incubated with these compounds in the absence (black bars) or presence (gray bars) of human neutrophils by calcofluor white quantification (data are mean +/−SD of five replicate wells) <b>E.</b> C57BL/6 mice were infected with <i>A. fumigatus</i> and at 0 and 6 h post-infection 13.4 µg of simvastatin (Sv), deferiprone (11.1 µg), deferroxamine (52.5 µg), Sv+ deferiprone, or Sv+ deferroxamine was applied topically to infected corneas and eyes were imaged at 24 h post-infection. <b>F.</b> Metamorph image analysis was used to quantify fungal dsRed expression and <b>G.</b> eyes were homogenized for CFU analysis. B–D: data are mean +/−SD of five replicate wells; F,G: data points represent individual corneas. All panels show representative data from one experiment except for panel G which shows pooled data from repeat experiments. Similar results were found in three repeat experiments. Abbreviations: <b>HMG</b>- 3-hydroxy-3-methyl-glutaryl-CoA, <b>Sv</b>-simvastatin, <b>Lv</b>-lovastatin, <b>Dprone</b>- deferiprone, <b>Defox</b>-deferroxamine.</p

Fungal strains utilized in this study.

<p>Fungal strains utilized in this study.</p

Expression of local and systemic iron-sequestration proteins in <i>Aspergillus fumigatus</i> infected corneas.

<p><b>A.</b> IL-6 production in corneas of C57BL/6 and Dectin-1^−/− mice 10 h after infection with <i>A. fumigatus</i>. <b>B.</b> Serum IL-6 at 24 h post-infection was quantified by ELISA <b>C.</b> Total liver hepcidin gene expression was quantified using qPCR 24 h after corneal infection. <b>D.</b> Total neutrophil numbers in corneas of C57BL/6 and IL-6^−/− mice 24 h post-infection. Neutrophils were incubated with the Ly6G NIMP-R14 Ab, and examined by flow cytometry. (Data points represent individual corneas) <b>E.</b> RNA was extracted from corneas of infected mice 24 h post-infection, and genes encoding proteins involved in iron chelation, heme or siderophore sequestration, and hepcidin signaling were examined by qPCR. <b>F.</b> Lcn-1 gene expression in peripheral blood neutrophils from healthy volunteers after 2 h incubation with crude hyphal extract (CHE). (Data in panels A, B, C and E are mean +/− SD of 5 mice per group; Data in panel F are from three separate human donors). Abbreviations: <b>B6</b>-C57BL/6, <b>CHE</b>- crude Aspergillus hyphal extract, <b>D1</b>-Dectin-1, <b>Lf</b>-lactoferrin, <b>Tf</b>-transferrin, <b>LR</b>- lactoferrin receptor, <b>TR</b>- transferrin receptor, <b>HptG</b>- haptoglobin, <b>Hpx</b>- hemopexin, <b>Lcn</b>-lipocalin, <b>HFE</b>- human hemochromatosis protein, <b>BMP</b>-bone morphogenetic protein, <b>Alk</b>-activin receptor-like kinase, <b>Actr</b>-actin-related protein, <b>HJV</b>- hemojuvelin, <b>HAMP</b>-hepcidin, <b>Fpt</b>- ferroportin.</p

The battle for iron between the mammalian host and fungi.

<p><b>Defox</b>- deferroxamine, <b>Dprone</b>- deferiprone, <b>Fe</b>-iron, <b>Fus C</b>- fusarinine C, <b>HMG-CoA</b>- 3-hydroxy-3methylglutaryl-coenzyme, <b>Lcn-1</b>- lipocalin 1, <b>Lf</b>- lactoferrin, <b>NOX</b>- nicotinamide adenine dinucleotide phosphate oxidase, <b>ROS</b>-reactive oxygen species, <b>TAFC</b>- triacetyl fusarinine C.</p

Effect of exogenous lipocalin-1 in <i>A. fumigatus</i> corneal infection.

<p><b>A.</b> Pathway showing Lcn-1 sequestration of fungal siderophores. <b>B.</b> Growth of <i>A. fumigatus</i> incubated with recombinant human Lcn-1 in the absence (black bars) or presence (gray bars) of human neutrophils determined by calcofluor white binding and quantification using fluorometry (data are mean +/−SD of five replicate wells). <b>C–E:</b> C57BL/6 mice were given topical Lcn-1 (16 µg) at 0 and 6 h post-infection with <i>A. fumigatus</i> dsRed. <b>C.</b> Representative corneas; <b>D.</b> Metamorph image analysis showing fungal dsRed expression and <b>E.</b> CFU per eye. Data points represent individual corneas. All panels show representative data from one experiment except for panel E which shows pooled data from repeat experiments. Similar results were found in two repeat experiments. Abbreviations: <b>FusC</b>- fusarinine C, <b>TAFC</b>- tri-acetyl fusarinine C, <b>Lcn</b>-lipocalin.</p

Role of mevalonate pathway for extracellular siderophores in <i>A. fumigatus</i> corneal infection.

<p><b>A.</b> Mevalonate extracellular siderophore biosynthesis pathway. <b>B–E.</b> C57BL/6 mice infected intrastromally with <i>A. fumigatus</i> Δ<i>sidH</i> and Δ<i>sidI</i> mutant strains. <b>B.</b> CFU per eye at 4 h and 48 h post-infection. <b>C.</b> Representative eyes showing corneal opacity <b>D.</b> Quantification of percent corneal opacity and <b>E.</b> total corneal opacity. B, D, E: Data points represent individual corneas. All panels show representative data from one experiment except for panel B which shows pooled data from repeat experiments. Similar results were found in 3 repeat experiments. Abbreviations: <b>HMG-CoA</b>: 3-hydroxy-3-methyl-glutaryl-CoA.</p

Susceptibility of <i>A. fumigatus</i> siderophore and iron acquisition mutants in fungal keratitis.

<p><b>A.</b> Growth <i>A. fumigatus</i> Δ<i>sidA</i> and Δ<i>hapX</i> mutants after incubation with human neutrophils; fungal mass was quantified using calcofluor white and fluorometry. (Mean +/− SD of 5 replicate wells.) <b>B–F: </b><i>A. fumigatus</i> corneal infection; <b>G–J </b><i>F. oxysporum</i> corneal infection. <b>B:</b> CFU per eye of C57BL/6 mice 48 h after intrastromal infection with <i>A. fumigatus</i> Δ<i>sidA</i>, Δ<i>hapX</i>, or Δ<i>ftrA</i> mutants. <b>C.</b> Representative eyes showing corneal opacification at 24 h and 48 h. <b>D.</b> Representative corneal sections stained with periodic acid-schiff and hematoxylin (PASH). <b>E.</b> Quantification of percent corneal opacity and <b>F.</b> Total cornea opacity. <b>G–J.</b> C57BL/6 corneas were infected with <i>F. oxysporum</i> Δ<i>hapX</i> and the parent strain. <b>G.</b> CFU per eye at 4 h and 48 h post-infection <b>H.</b> Representative eyes showing corneal opacification. <b>I.</b> Quantification of percent corneal opacity <b>J.</b> Total cornea opacity. Data points represent individual corneas. All panels show representative data from one experiment except for panels B and G which show pooled data from repeat experiments. Similar results were found in three repeat experiments. Abbreviations: <b>Neuts</b>- neutrophils, <b>epi</b>-epithelium, <b>endo</b>-endothelium, <b>AC</b>-anterior chamber.</p