Protein expression of <i>S. pneumoniae</i> pneumolysin and <i>P. aeruginosa</i> exotoxins.

<p>A. Western blot of pneumolysin in the ATCC reference strain (Lane 1), and four representative clinical isolates (lanes 2–5). B. Western blot of culture supernatants from <i>Pseudomonas aerugenosa</i> reference strains PAO1, which expresses ExoS and ExoT, and PA103, which expresses ExoU and ExoT. C. ExoS, ExoT and ExoU expression in representative <i>Pseudomonas</i> clinical isolates. Lane 1 is similar to PAO1 in expressing ExoS and ExoT; Lane 2 is similar to PA103 in expressing ExoU and ExoT; Lane 3 is <i>P. otitidis</i>, which does not express Type III exotoxins; Lane-4 Clinical isolate express all three effector molecules; Lane-5 Exo U and Exo T expressing clinical isolate similar to PA103. D. percent and total exotoxin production by 49 clinical isolates.</p

Cellular composition of corneal ulcers from patients with bacterial keratitis.

<p>Representative corneal ulcers of patients caused by <i>P. aeruginosa</i> (A), or by <i>S. pneumoniae</i> (B). C, D. Gram staining of corneal ulcer material showing Gram negative bacilli (C), and Gram positive diplococci and chains (D). Original magnification is x1000. E,F: Wrights Giemsa (Diff-Quik) stain of corneal ulcer material from <i>P. aeruginosa</i> (E), or <i>S. pneumoniae</i> (F) infected tissue Original magnification is x400. G. Percent neutrophils and mononuclear cells were determined by counting cells from ten <i>P. aeruginosa</i> and ten <i>S. pneumoniae</i> patients.</p

Primer sequences.

<p>Primer sequences.</p

Clinical characteristics.

<p>Data are number and percent (%) of patients, unless otherwise indicated. Patients (n = 48) had corneal ulcers and presented at the clinic within 1–2 weeks after infection; corneal scrapings from the ulcer were used in the present study. Ten donor corneas from individuals with no infection or inflammation were obtained from the International Rotary Aravind Eye Bank. NA, not applicable.</p>*<p>accumulation of neutrophils in the anterior chamber.</p

Gene expression of Toll Like Receptors, inflammasome proteins and cytokines in corneal ulcers from patients with bacterial keratitis.

<p>RNA was extracted from corneal ulcers, reverse transcribed and processed for Q-PCR. Data points represent individual patients infected with <i>P. aeruginosa</i> (closed circles) or <i>S. pneumoniae</i> (open circles), and the values presented are the log of relative gene expression (log(RQ)) in relation to uninfected donor corneas calculated using the 2^−ΔΔct method described in Methods. A. Pro-inflammatory cytokines IL-1α, IL-1β and IFN-γ; B. Toll Like Receptors and C. Inflammasome proteins. There were no significant differences in gene expression between <i>P. aeruginosa</i> and <i>S. pneumoniae</i> (p>0.05).</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

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

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

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