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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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ΔpacC mutants are less avidly internalised by A549 and spore internalisation depends on Dectin-1.(A) Percent internalisation by nystatin protection assay. Epithelial monolayers were incubated with 106 spores/ml for 4 hr, n  =  9 for wild types and ΔpacC. (B) Percent internalisation, when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or actin-mediated internalisation (0.2 µM CD), or in the presence of an isotype control antibody (0.3 µg/ml) technical and biological duplicates. (C) Percent detachment, relative to PBS challenge, of A549 upon infection with A. fumigatus (105 spores/ml) for 16 hr and inhibition of actin-mediated internalisation via Cytochalasin D (CD, 0.2 µM), when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or in the presence of an isotype control antibody (0.3 µg/ml), technical and biological triplicates. (D) Use of a soluble Fc Dectin-1 protein for immunofluorescence-mediated imaging of β-glucan in A. fumigatus spores, 4 hr, supplemented DMEM. (E)A. fumigatus spore size (μM2), 4 hr, supplemented DMEM. A, B, C: unpaired t test (unless otherwise indicated) *** p<0.001, ** 0.001<p<0.01, and * 0.01<p<0.05.
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ppat-1004413-g006: ΔpacC mutants are less avidly internalised by A549 and spore internalisation depends on Dectin-1.(A) Percent internalisation by nystatin protection assay. Epithelial monolayers were incubated with 106 spores/ml for 4 hr, n  =  9 for wild types and ΔpacC. (B) Percent internalisation, when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or actin-mediated internalisation (0.2 µM CD), or in the presence of an isotype control antibody (0.3 µg/ml) technical and biological duplicates. (C) Percent detachment, relative to PBS challenge, of A549 upon infection with A. fumigatus (105 spores/ml) for 16 hr and inhibition of actin-mediated internalisation via Cytochalasin D (CD, 0.2 µM), when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or in the presence of an isotype control antibody (0.3 µg/ml), technical and biological triplicates. (D) Use of a soluble Fc Dectin-1 protein for immunofluorescence-mediated imaging of β-glucan in A. fumigatus spores, 4 hr, supplemented DMEM. (E)A. fumigatus spore size (μM2), 4 hr, supplemented DMEM. A, B, C: unpaired t test (unless otherwise indicated) *** p<0.001, ** 0.001<p<0.01, and * 0.01<p<0.05.

Mentions: Epithelial cells have been shown to internalise and kill up to 50% of the A. fumigatus spores they come into contact with [16]–[19], [22]. We therefore hypothesised that contact-dependent perturbations of epithelial integrity might result from internalisation of fungal spores. To assess this we first assessed the numbers of internalised wild type and ΔpacC spores using a nystatin protection assay (Figure 6A). This revealed that the proportion of wild-type spores internalised by A549 cells ranges from 16 to 23% (Figure 6A). However, epithelial cells were found to internalise ΔpacC mutants significantly less avidly than the respective parental isolates (Figure 6A), whereby only ∼10–12% of the initial inoculum had become internalised after 4 hours of co-incubation. At the concentrations used in this assay nystatin exposure was 100% efficient in killing A. fumigatus spores (Figure S11).


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)

ΔpacC mutants are less avidly internalised by A549 and spore internalisation depends on Dectin-1.(A) Percent internalisation by nystatin protection assay. Epithelial monolayers were incubated with 106 spores/ml for 4 hr, n  =  9 for wild types and ΔpacC. (B) Percent internalisation, when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or actin-mediated internalisation (0.2 µM CD), or in the presence of an isotype control antibody (0.3 µg/ml) technical and biological duplicates. (C) Percent detachment, relative to PBS challenge, of A549 upon infection with A. fumigatus (105 spores/ml) for 16 hr and inhibition of actin-mediated internalisation via Cytochalasin D (CD, 0.2 µM), when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or in the presence of an isotype control antibody (0.3 µg/ml), technical and biological triplicates. (D) Use of a soluble Fc Dectin-1 protein for immunofluorescence-mediated imaging of β-glucan in A. fumigatus spores, 4 hr, supplemented DMEM. (E)A. fumigatus spore size (μM2), 4 hr, supplemented DMEM. A, B, C: unpaired t test (unless otherwise indicated) *** p<0.001, ** 0.001<p<0.01, and * 0.01<p<0.05.
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ppat-1004413-g006: ΔpacC mutants are less avidly internalised by A549 and spore internalisation depends on Dectin-1.(A) Percent internalisation by nystatin protection assay. Epithelial monolayers were incubated with 106 spores/ml for 4 hr, n  =  9 for wild types and ΔpacC. (B) Percent internalisation, when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or actin-mediated internalisation (0.2 µM CD), or in the presence of an isotype control antibody (0.3 µg/ml) technical and biological duplicates. (C) Percent detachment, relative to PBS challenge, of A549 upon infection with A. fumigatus (105 spores/ml) for 16 hr and inhibition of actin-mediated internalisation via Cytochalasin D (CD, 0.2 µM), when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or in the presence of an isotype control antibody (0.3 µg/ml), technical and biological triplicates. (D) Use of a soluble Fc Dectin-1 protein for immunofluorescence-mediated imaging of β-glucan in A. fumigatus spores, 4 hr, supplemented DMEM. (E)A. fumigatus spore size (μM2), 4 hr, supplemented DMEM. A, B, C: unpaired t test (unless otherwise indicated) *** p<0.001, ** 0.001<p<0.01, and * 0.01<p<0.05.
Mentions: Epithelial cells have been shown to internalise and kill up to 50% of the A. fumigatus spores they come into contact with [16]–[19], [22]. We therefore hypothesised that contact-dependent perturbations of epithelial integrity might result from internalisation of fungal spores. To assess this we first assessed the numbers of internalised wild type and ΔpacC spores using a nystatin protection assay (Figure 6A). This revealed that the proportion of wild-type spores internalised by A549 cells ranges from 16 to 23% (Figure 6A). However, epithelial cells were found to internalise ΔpacC mutants significantly less avidly than the respective parental isolates (Figure 6A), whereby only ∼10–12% of the initial inoculum had become internalised after 4 hours of co-incubation. At the concentrations used in this assay nystatin exposure was 100% efficient in killing A. fumigatus spores (Figure S11).

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