1. Volume 19, Issue 1, Pages 79-90 (January 2016)
Aspergillus Cell Wall Melanin Blocks LC3-Associated Phagocytosis to Promote Pathogenicity 
Tonia Akoumianaki, Irene Kyrmizi, Isabel Valsecchi, Mark S. Gresnigt, George Samonis, Elias Drakos, Dimitrios Boumpas, Laetitia Muszkieta, Marie-Christine Prevost, Dimitrios P. Kontoyiannis, Triantafyllos Chavakis, Mihai G. Netea, Frank L. van de Veerdonk, Axel A. Brakhage, Jamel El-Benna, Anne Beauvais, Jean-Paul Latge, Georgios Chamilos 
Cell Host & Microbe 
Volume 19, Issue 1, Pages (January 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 19, 79-90DOI: (10.1016/j.chom.2015.12.002)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
A. fumigatus Cell Wall Melanin Removal Is Required for Activation of LAP
(A–C) Primary human monocytes were infected at different time points with FITC-labeled live conidia of the indicated A. fumigatus strain. Cells were stained for LC3 (red) and TOPRO-3 (blue, nuclear staining) and analyzed by confocal microscopy. Data on quantification of LC3+ phagosomes are presented as mean ± SEM of three independent experiments.
(D and E) Primary human monocytes were infected with FITC-labeled PFA-killed conidia as in (A) and analyzed by confocal microscopy. Representative immunofluorescence images are shown.
(F) Phagosomes from primary human monocytes infected for 30 min with the indicated A. fumigatus strains were isolated, equal amounts of phagosome protein extracts (15 μg) were prepared and levels of LC3 protein were determined by immunoblotting. Actin was used as a loading control. Representative Immunoblotting of one out of three independent experiments is shown. ∗∗p < , ∗p < 0.01, paired Student’s t test. Scale bar, 5 μm (see also related Figures S1–S3).
Cell Host & Microbe  , 79-90DOI: ( /j.chom )
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4. Figure 2
Purified Synthetic Melanin Inhibits Activation of LAP by A. fumigatus
(A) Levels of β-glucan surface expression in dormant conidia of Ku80, ΔrodA/pksP, or ΔrodA/pksP coated with purified synthetic melanin (melanin-coated ΔrodA/pksP) following immunostaining with the use of Alexa555 secondary antibody were determined by measurement of relative fluorescent intensity at the FL1 channel (log MFI), and representative FL1 histograms are shown.
(B and C) Data on quantification of LC3+ phagosomes in primary human monocytes infected with the indicated A. fumigatus strain are presented as mean ± SEM of three independent experiments. Representative immunofluorescence images are shown. Scale bar, 5 μm.
(D) Primary human monocytes were stimulated for 1 hr with ΔpksP with or without increasing concentrations of purified synthetic melanin added at 15 min of infection, and LC3 protein levels in cell lysates were determined by immunoblotting. Actin was used as a loading control.
(E) Data on quantification of LC3+ phagosomes in primary human monocytes stimulated as in (D) are presented as mean ± SEM of three independent experiments. ∗∗p < , paired Student’s t test.
Cell Host & Microbe  , 79-90DOI: ( /j.chom )
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5. Figure 3
LAP Regulates Phagosome Biogenesis and Killing of melanin-Deficient ΔpksP A. fumigatus
(A) PMA-differentiated THP-1 cells preloaded with LysoTracker Red were infected for 2 hr with FITC-labeled conidia of the indicated A. fumigatus strain and immediately analyzed by confocal microscopy. Data on quantification of Lysotracker+ phagosomes are presented as mean ± SEM from one out of three independent experiments. Representative immunofluorescence images are shown. Scale bar, 5 μm.
(B) PMA-differentiated THP-1 cells were infected with FITC-labeled conidia of the indicated A. fumigatus strain at a MOI of 1:1, intracellular conidia were harvested and the percentage of killed (PI+) conidia at 2 hr and 6 hr of infection was immediately assessed by confocal imaging. Data are expressed as mean ± SEM of three independent experiments.
(C) Levels of Atg5 protein determined by immunoblotting in cell lysates of THP-1 cells at 48 hr of transfection by Amaxa electroporation with a pool of RNAi sequences targeting ATG5 versus scramble control RNAi (C RNAi). Actin was used as loading control for immunoblot.
(D) Data on quantification of Lysotracker+ phagosomes in PMA-differentiated THP-1 cells transfected as described in (C) and 48 hr later infected with FITC-labeled conidia of ΔpksP are presented as mean ± SEM of one out of three independent experiments. Representative immunofluorescence images are shown. Scale bar, 5 μm.
(E) THP-1 cells transfected and infected as in D. Intracellular conidia were harvested at 2 hr and 6 hr of infection, the percentage of killed (PI+) conidia was calculated as described in (B), and data are expressed as mean ± SEM of three independent experiments.
(F) GFP-LC3b BMDMs were infected at different time points with Alexa633-labeled conidia of the indicated A. fumigatus strains and analyzed by confocal microscopy. Data on quantification of GFP-LC3+ A. fumigatus-containing phagosomes are presented as mean ± SEM of three independent experiments. Representative immunofluorescence images are shown. Scale bar, 10 μm.
(G) BMDMs from control (Atg5flox/flox) and Atg5 conditional KO (Atg5 flox/flox +vavCre) mice were infected with conidia of ΔpksP A. fumigatus and the percentage of conidial killing was calculated as described in (B). Data are expressed as mean ± SEM of three independent experiments; ∗∗p < , ∗p < 0.01, paired Student’s t test (see also related Figures S4 and S5).
Cell Host & Microbe  , 79-90DOI: ( /j.chom )
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6. Figure 4 Autophagy Regulates A. fumigatus Virulence In Vivo
(A) Fungal load in lungs of Atg5 conditional KO (Atg5 flox/flox +vavCre) and control (Atg5 flox/− +vavCre) mice challenged with a sublethal dose of either wild-type ATCC46645 or ΔpksP conidia and 14 days later reinfected with 5 × 107 conidia of the same A. fumigatus strain used in the first challenge following immunosuppression with cyclophosphamide. Lungs were removed and homogenized on day 2 of infection and CFU counts were assessed. ∗∗p < ; ∗p < 0. 01, unpaired Student’s t test.
(B) Survival of control (Atg5 flox/flox, n = 8) and Atg5 conditional KO (Atg5 flox/flox +vavCre, n = 6) mice infected with ΔpksP conidia as in (A). p < 0.01, for control versus Atg5 conditional KO mice, log-rank (Mantel-Cox) test.
(C) Lungs of representative control (Atg5 flox/flox) and Atg5 conditional KO (Atg5 flox/flox +vavCre) mice, cyclophosphamide-immunosuppressed and reinfected with ΔpksP conidia as in (A) or uninfected and sacrificed on day 2 of infection.
(D), Representative photomicrographs of fungal growth in periodic acid Schiff-stained lungs from (C). Original magnification 400×. Arrowhead shows an area with characteristic tissue invasion by A. fumigatus hyphae.
(E) In vivo killing of wild-type (ATCCC 46645) and ΔpksP conidia by pulmonary phagocytes recovered by BAL from non-immunosuppressed Atg5 conditional KO (Atg5 flox/flox +vavCre; n = 3 per group) and control (Atg5 flox/flox, n = 3 per group) mice at 24 hr of intratracheal nonlethal infection with 107 conidia. ∗∗p < ; ∗p < 0.01, unpaired Student’s t test.
(F) Fungal load in lungs of non-immunosuppressed Atg5 conditional KO (Atg5flox/flox+ vavCre) and control (Atg5flox/flox) mice infected as in (E). Lungs fungal loads on day 1 of infection were assessed as in (A). ∗∗p < , unpaired Student’s t test.
Cell Host & Microbe  , 79-90DOI: ( /j.chom )
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7. Figure 5
A. fumigatus Melanin Selectively Inhibits NADPH Oxidase-Dependent Activation of LAP
(A) TNF and IL-1β production in culture supernatants of primary human monocytes left untreated or stimulated overnight with PFA-inactivated conidia of the indicated A. fumigatus strains. Data are presented as mean ± SEM of three independent experiments.
(B) IL-1β production in culture supernatants of primary human monocytes stimulated as in (A) or stimulated with LPS plus ATP added for the last 20 min of incubation, with or without pretreatment for 30 min with Syk inhibitor (574711, Calbiochem, 1 μM).
(C) Intracellular ROS production in primary human monocytes left untreated (gray solid area), or infected with the indicated A. fumigatus strains (black solid line) were determined by measurement of relative fluorescent intensity of DCFH-DA at the FL1 channel (log MFI).
(D) Differences in intracellular ROS production between experimental groups were quantified and data are presented as mean ± SEM from four independent experiments.
(E) Intracellular ROS production measured by DCFH-DA in primary human monocytes infected with conidia of ΔpksP A. fumigatus with or without pretreatment with NAC. Representative FL1 histograms are shown.
(F) Data on quantification of LC3+ phagosomes in primary human monocytes infected with conidia of ΔpksP A. fumigatus with or without pretreatment with NAC, presented as mean ± SEM of one out of three independent experiments.
(G and H) Data on quantification of LC3+ phagosomes in monocytes of CGD patients and healthy controls (HC) infected with melanin-deficient A. fumigatus strains, are presented as mean ± SEM of one out of three independent experiments. ∗∗p < , ∗p < 0.01, paired Student’s t test.
Cell Host & Microbe  , 79-90DOI: ( /j.chom )
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8. Figure 6
A. fumigatus Melanin Excludes p22phox Subunit from the Phagosome Membrane
(A and B) Primary human monocytes were infected for 30 min with FITC-labeled conidia of the indicated A. fumigatus strains and stained for p47phox. Data on quantification of p47phox+ phagosomes are presented as mean ± SEM of three independent experiments. Representative immunofluorescence images are shown. Scale bar, 5 μm.
(C) Primary human monocytes were stimulated at different time points as in (A), and phosphorylation of p47phox (p-Ser345-phox47) was determined in cell lysates by immunoblot analysis. Actin and p47phox were used as loading controls.
(D) Representative immunofluorescence images of primary human monocytes infected as in (A) and stained for p22phox.
(E) Data on quantification of p22phox+ phagosomes are presented as mean ± SEM of three independent experiments. Scale bar, 5 μm.
(F) Primary human monocytes were stimulated for 15 min with peroxide-treated (melanin bleaching) or untreated conidia of either ΔrodA or ΔrodA/pksP, fixed and stained for p22phox as in (D). Data on quantification of p22phox+ phagosomes are presented as mean ± SEM of three independent experiments.
(G) Primary human monocytes were stimulated with the indicated A. fumigatus strains following 30 min pretreatment with 20 mM NAC, fixed and stained as in (D). Data on quantification of p22phox+ phagosomes are presented as mean ± SEM of three independent experiments.
(H) Primary human monocytes were left untreated or stimulated with A. fumigatus melanin or purified synthetic melanin for 1 hr at 37°C. Glucose oxidase (GO, 2 μg/ml) and DCFH-DA were added for 30 min and intracellular ROS production was determined as shown in representative FL1 histograms.
(I) Primary human monocytes were stimulated for 15 min with purified A. fumigatus ATCC melanin particles (melanin ghosts) or melanin-deficient Δpksp ghosts, fixed, and stained for p22phox as in (D).
(J) Data on quantification of p22phox+ phagosomes are presented as mean ± SEM of three independent experiments. ∗∗p < , paired Student’s t test (see also related Figures S6 and S7).
Cell Host & Microbe  , 79-90DOI: ( /j.chom )
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9. Figure 7
Aspergillus nidulans Melanin Inhibits NADPH Oxidase-Dependent Activation of LAP
(A) Killing of melanin-deficient (wA) or the parental wild-type (wt) strain of A. nidulans by primary human monocytes, assessed by CFU counts. Data are representative of three independent experiments; error bars represent the SD of quadruplicate wells.
(B) Intracellular ROS production in primary human monocytes infected with the indicated A. nidulans strain assessed by DCFH-DA relative fluorescent intensity at the FL1 channel (log MFI).
(C) Differences in ROS production between experimental groups were quantified and data are presented as mean ± SEM from three independent experiments.
(D) Representative images of p22phox immunostaining in primary human monocytes stimulated with FITC-labeled conidia of the indicated A. nidulans strain. Scale bar, 10 μm.
(E) Data on quantification of p22phox+ phagosomes are presented as mean ± SEM of three independent experiments.
(F) Representative images of LC3 immunostaining in primary human monocytes stimulated as in (D). Scale bar, 10 μm.
(G) Data on quantification of LC3+ phagosomes are presented as mean ± SEM of three independent experiments. ∗∗p < , ∗p < 0.01, paired Student’s t test.
Cell Host & Microbe  , 79-90DOI: ( /j.chom )
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