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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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PacC is required for pathogenicity and epithelial invasion in leukopenic mice.(A) Kaplan-Meier curve for murine survival (n ≥ 9) after infection with 6 × 105 and 5 × 104 spores for ATCC46645 and CEA10 respectively. (B) Histopathology of leukopenic murine lungs after 4, 8, 12, hr of infection with ATCC46645 and ΔpacCATCC (108 spores), Grocott's Methenamine silver (GMS) and light green staining, 200× magnification. (C)ΔpacCATCC hyphae (black arrows) are unable to penetrate the respiratory epithelium (RE), BA indicates bronchial airway space, 20 hr post-infection, 400× magnification.
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ppat-1004413-g002: PacC is required for pathogenicity and epithelial invasion in leukopenic mice.(A) Kaplan-Meier curve for murine survival (n ≥ 9) after infection with 6 × 105 and 5 × 104 spores for ATCC46645 and CEA10 respectively. (B) Histopathology of leukopenic murine lungs after 4, 8, 12, hr of infection with ATCC46645 and ΔpacCATCC (108 spores), Grocott's Methenamine silver (GMS) and light green staining, 200× magnification. (C)ΔpacCATCC hyphae (black arrows) are unable to penetrate the respiratory epithelium (RE), BA indicates bronchial airway space, 20 hr post-infection, 400× magnification.

Mentions: Leukopenia is an important risk factor for IA in humans, and cyclophosphamide-induced leukocyte depletion renders mice highly susceptible to pulmonary infection [34]. To assess the role of A. fumigatus PacC in pathogenicity we assessed the survival of leukopenic mice following infection, via the intranasal route, with spores of wild-type, ΔpacC or reconstituted isolates (Figure 2A). Relative to wild-type strains, ΔpacCATCC and ΔpacCCEA10 mutants were significantly attenuated for virulence (Figure 2A). At day 6 post-infection 100% of mice infected with the ΔpacC mutants remained alive while 78% and 92% of mice infected with wild type (ATCC46645 and CEA10 respectively) isolates were dead.


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)

PacC is required for pathogenicity and epithelial invasion in leukopenic mice.(A) Kaplan-Meier curve for murine survival (n ≥ 9) after infection with 6 × 105 and 5 × 104 spores for ATCC46645 and CEA10 respectively. (B) Histopathology of leukopenic murine lungs after 4, 8, 12, hr of infection with ATCC46645 and ΔpacCATCC (108 spores), Grocott's Methenamine silver (GMS) and light green staining, 200× magnification. (C)ΔpacCATCC hyphae (black arrows) are unable to penetrate the respiratory epithelium (RE), BA indicates bronchial airway space, 20 hr post-infection, 400× magnification.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4199764&req=5

ppat-1004413-g002: PacC is required for pathogenicity and epithelial invasion in leukopenic mice.(A) Kaplan-Meier curve for murine survival (n ≥ 9) after infection with 6 × 105 and 5 × 104 spores for ATCC46645 and CEA10 respectively. (B) Histopathology of leukopenic murine lungs after 4, 8, 12, hr of infection with ATCC46645 and ΔpacCATCC (108 spores), Grocott's Methenamine silver (GMS) and light green staining, 200× magnification. (C)ΔpacCATCC hyphae (black arrows) are unable to penetrate the respiratory epithelium (RE), BA indicates bronchial airway space, 20 hr post-infection, 400× magnification.
Mentions: Leukopenia is an important risk factor for IA in humans, and cyclophosphamide-induced leukocyte depletion renders mice highly susceptible to pulmonary infection [34]. To assess the role of A. fumigatus PacC in pathogenicity we assessed the survival of leukopenic mice following infection, via the intranasal route, with spores of wild-type, ΔpacC or reconstituted isolates (Figure 2A). Relative to wild-type strains, ΔpacCATCC and ΔpacCCEA10 mutants were significantly attenuated for virulence (Figure 2A). At day 6 post-infection 100% of mice infected with the ΔpacC mutants remained alive while 78% and 92% of mice infected with wild type (ATCC46645 and CEA10 respectively) isolates were dead.

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