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The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

Bertuzzi M, Schrettl M, Alcazar-Fuoli L, Cairns TC, Muñoz A, Walker LA, Herbst S, Safari M, Cheverton AM, Chen D, Liu H, Saijo S, Fedorova ND, Armstrong-James D, Munro CA, Read ND, Filler SG, Espeso EA, Nierman WC, Haas H, Bignell EM - PLoS Pathog. (2014)

Bottom Line: We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection.Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences.Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

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Internalisation and Dectin-1 protect murine pulmonary epithelia from A. fumigatus-mediated damage.(A) Histopathology of Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected with CEA10 (108 spores, 24 hr of infection, GMS and light green staining, 200× magnification). (B) LDH/BCA fold change in BALs from Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected as in B, technical triplicates. (C) S100B western blotting on lung homogenates from Dectin-1−/− and Dectin-1+/+ leukopenic mice infected as in B. Densitometry plot where pooled densitometry values for S100B were normalised on pooled densitometry values for actin and background subtracted for the saline controls. (D) Macrophages and neutrophils (cells/ml) in BALs from immunocompetent mice infected with ΔpacCCEA10 mutant and CEA10 (n  =  4, 106 spores, 24 hr of infection). B and C: unpaired t test, *** p<0.001 and * 0.01<p<0.05.
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ppat-1004413-g007: Internalisation and Dectin-1 protect murine pulmonary epithelia from A. fumigatus-mediated damage.(A) Histopathology of Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected with CEA10 (108 spores, 24 hr of infection, GMS and light green staining, 200× magnification). (B) LDH/BCA fold change in BALs from Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected as in B, technical triplicates. (C) S100B western blotting on lung homogenates from Dectin-1−/− and Dectin-1+/+ leukopenic mice infected as in B. Densitometry plot where pooled densitometry values for S100B were normalised on pooled densitometry values for actin and background subtracted for the saline controls. (D) Macrophages and neutrophils (cells/ml) in BALs from immunocompetent mice infected with ΔpacCCEA10 mutant and CEA10 (n  =  4, 106 spores, 24 hr of infection). B and C: unpaired t test, *** p<0.001 and * 0.01<p<0.05.

Mentions: The extent of epithelial protection afforded by in vitro delivery of Mab1859 (Figures 6B and C) was suggestive of a detrimental role for Dectin-1 engagement during A. fumigatus-epithelial interactions. To decipher between protective and exacerbatory roles for Dectin-1 in maintenance of epithelial integrity in whole animals, we assessed pulmonary damage after 24 hours of A. fumigatus infection in Dectin-1+/+ and Dectin-1−/− mice. To study epithelial activities in the absence of confounding leukocyte responses, mice were depleted of leukocytes using a cyclophosphamide and hydrocortisone protocol and lung injury was scored via histological, biochemical and immunoblot assays. In the lungs of Dectin-1+/+ and Dectin-1−/− mice, fungal lesions were equivalent in size and invasiveness (Figure 7A) although frequency of fungal lesions was increased in Dectin-1−/− animals (not shown). Epithelial damage was surveyed via quantitation of lactate dehydrogenase (LDH) in BALs (Figure 7B) and analysis of expression of the Dectin-1-independent damage associated molecular pattern (DAMP) protein S100B (Figure 7C), whose major source during A. fumigatus infection is epithelial cells [48]. Both assays revealed heightened epithelial damage in the lungs of Dectin-1−/− animals relative to wild type counterparts. Our results indicate that, despite a highly protective role for the anti Dectin-1 antibody Mab1859 during in vitro epithelial infections, integrity of Dectin-1 (Figures 7A–C) is essential for limitation of epithelial damage in vivo. As neutrophil-depletion and macrophage dysfunction were chemotherapeutically implemented in our murine model we conclude that Dectin-1 activity is essential for protecting the lung epithelium from the damage inflicted by germinating A. fumigatus spores.


The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

Bertuzzi M, Schrettl M, Alcazar-Fuoli L, Cairns TC, Muñoz A, Walker LA, Herbst S, Safari M, Cheverton AM, Chen D, Liu H, Saijo S, Fedorova ND, Armstrong-James D, Munro CA, Read ND, Filler SG, Espeso EA, Nierman WC, Haas H, Bignell EM - PLoS Pathog. (2014)

Internalisation and Dectin-1 protect murine pulmonary epithelia from A. fumigatus-mediated damage.(A) Histopathology of Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected with CEA10 (108 spores, 24 hr of infection, GMS and light green staining, 200× magnification). (B) LDH/BCA fold change in BALs from Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected as in B, technical triplicates. (C) S100B western blotting on lung homogenates from Dectin-1−/− and Dectin-1+/+ leukopenic mice infected as in B. Densitometry plot where pooled densitometry values for S100B were normalised on pooled densitometry values for actin and background subtracted for the saline controls. (D) Macrophages and neutrophils (cells/ml) in BALs from immunocompetent mice infected with ΔpacCCEA10 mutant and CEA10 (n  =  4, 106 spores, 24 hr of infection). B and C: unpaired t test, *** p<0.001 and * 0.01<p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199764&req=5

ppat-1004413-g007: Internalisation and Dectin-1 protect murine pulmonary epithelia from A. fumigatus-mediated damage.(A) Histopathology of Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected with CEA10 (108 spores, 24 hr of infection, GMS and light green staining, 200× magnification). (B) LDH/BCA fold change in BALs from Dectin-1−/− and Dectin-1+/+ leukopenic mice (n  =  3) infected as in B, technical triplicates. (C) S100B western blotting on lung homogenates from Dectin-1−/− and Dectin-1+/+ leukopenic mice infected as in B. Densitometry plot where pooled densitometry values for S100B were normalised on pooled densitometry values for actin and background subtracted for the saline controls. (D) Macrophages and neutrophils (cells/ml) in BALs from immunocompetent mice infected with ΔpacCCEA10 mutant and CEA10 (n  =  4, 106 spores, 24 hr of infection). B and C: unpaired t test, *** p<0.001 and * 0.01<p<0.05.
Mentions: The extent of epithelial protection afforded by in vitro delivery of Mab1859 (Figures 6B and C) was suggestive of a detrimental role for Dectin-1 engagement during A. fumigatus-epithelial interactions. To decipher between protective and exacerbatory roles for Dectin-1 in maintenance of epithelial integrity in whole animals, we assessed pulmonary damage after 24 hours of A. fumigatus infection in Dectin-1+/+ and Dectin-1−/− mice. To study epithelial activities in the absence of confounding leukocyte responses, mice were depleted of leukocytes using a cyclophosphamide and hydrocortisone protocol and lung injury was scored via histological, biochemical and immunoblot assays. In the lungs of Dectin-1+/+ and Dectin-1−/− mice, fungal lesions were equivalent in size and invasiveness (Figure 7A) although frequency of fungal lesions was increased in Dectin-1−/− animals (not shown). Epithelial damage was surveyed via quantitation of lactate dehydrogenase (LDH) in BALs (Figure 7B) and analysis of expression of the Dectin-1-independent damage associated molecular pattern (DAMP) protein S100B (Figure 7C), whose major source during A. fumigatus infection is epithelial cells [48]. Both assays revealed heightened epithelial damage in the lungs of Dectin-1−/− animals relative to wild type counterparts. Our results indicate that, despite a highly protective role for the anti Dectin-1 antibody Mab1859 during in vitro epithelial infections, integrity of Dectin-1 (Figures 7A–C) is essential for limitation of epithelial damage in vivo. As neutrophil-depletion and macrophage dysfunction were chemotherapeutically implemented in our murine model we conclude that Dectin-1 activity is essential for protecting the lung epithelium from the damage inflicted by germinating A. fumigatus spores.

Bottom Line: We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection.Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences.Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

Show MeSH
Related in: MedlinePlus