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

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

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

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