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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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Impact of pacC deletion upon A. fumigatus growth.(A and B) Colonial growth phenotypes on supplemented DMEM, 48 hr of growth, 103 conidia. (C) Immunofluorescence-mediated visualisation of A. fumigatus growth in co-culture with A549 epithelial cells, 16 hours, supplemented DMEM pH 7.4. Fungal cells are labelled with calcoflour white and host cells are labelled with FITC-conjugated concanavalin A, 105 spores/ml, 200× magnification. (D) Quantitation of cell size (μM2) at 16 hr of co-culture with A549 cells, 104 spores/ml, growth conditions as for panel C. (E) Branching frequency of A. fumigatus hyphae, 104 spores/ml, growth conditions as for panel C.
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ppat-1004413-g001: Impact of pacC deletion upon A. fumigatus growth.(A and B) Colonial growth phenotypes on supplemented DMEM, 48 hr of growth, 103 conidia. (C) Immunofluorescence-mediated visualisation of A. fumigatus growth in co-culture with A549 epithelial cells, 16 hours, supplemented DMEM pH 7.4. Fungal cells are labelled with calcoflour white and host cells are labelled with FITC-conjugated concanavalin A, 105 spores/ml, 200× magnification. (D) Quantitation of cell size (μM2) at 16 hr of co-culture with A549 cells, 104 spores/ml, growth conditions as for panel C. (E) Branching frequency of A. fumigatus hyphae, 104 spores/ml, growth conditions as for panel C.

Mentions: To characterise the role of the A. fumigatus PacC transcription factor (AFUA_3G11970) in pathogenicity we constructed and complemented alleles in two distinct A. fumigatus clinical isolates CEA10 and ATCC46645 (Figure S1). Relative to non-mutated isolates, PacC mutants assumed a compact colonial phenotype on supplemented solid DMEM medium pH 7.4 (Figure 1A) which, in contrast to colonies of wild type isolates, lacked peripheral invasive hyphae, and were composed of a denser hyphal network indicative of a hyperbranching morphology (Figure 1B). This compact morphology was pH-independent, being also observed in colonies grown on Aspergillus complete medium pH 6.5 (Figure S2A), where sporulation and pigmentation of ΔpacC mutants was equivalent to that of the wild type. To assess pH sensitivity of the ΔpacC mutants we examined, on pH-buffered minimal media, the extent of radial growth at pHs 8.0 and 7.2, relative to growth at pH 6.5 (Figures S2B and S2C respectively). Consistent with a role for PacC in alkaline adaptation, ΔpacC isolates suffered growth impairment at pH 8.0, achieving approximately 10–20% of the radial growth attained at pH 6.5 (Figure S2B), compared with 40% achieved by wild-type and reconstituted strains. However, sensitivity of ΔpacC isolates to growth at pH 7.2, which approximates the pH of the mammalian lung, did not differ from that of the wild type isolates (Figure S2C).


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)

Impact of pacC deletion upon A. fumigatus growth.(A and B) Colonial growth phenotypes on supplemented DMEM, 48 hr of growth, 103 conidia. (C) Immunofluorescence-mediated visualisation of A. fumigatus growth in co-culture with A549 epithelial cells, 16 hours, supplemented DMEM pH 7.4. Fungal cells are labelled with calcoflour white and host cells are labelled with FITC-conjugated concanavalin A, 105 spores/ml, 200× magnification. (D) Quantitation of cell size (μM2) at 16 hr of co-culture with A549 cells, 104 spores/ml, growth conditions as for panel C. (E) Branching frequency of A. fumigatus hyphae, 104 spores/ml, growth conditions as for panel C.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004413-g001: Impact of pacC deletion upon A. fumigatus growth.(A and B) Colonial growth phenotypes on supplemented DMEM, 48 hr of growth, 103 conidia. (C) Immunofluorescence-mediated visualisation of A. fumigatus growth in co-culture with A549 epithelial cells, 16 hours, supplemented DMEM pH 7.4. Fungal cells are labelled with calcoflour white and host cells are labelled with FITC-conjugated concanavalin A, 105 spores/ml, 200× magnification. (D) Quantitation of cell size (μM2) at 16 hr of co-culture with A549 cells, 104 spores/ml, growth conditions as for panel C. (E) Branching frequency of A. fumigatus hyphae, 104 spores/ml, growth conditions as for panel C.
Mentions: To characterise the role of the A. fumigatus PacC transcription factor (AFUA_3G11970) in pathogenicity we constructed and complemented alleles in two distinct A. fumigatus clinical isolates CEA10 and ATCC46645 (Figure S1). Relative to non-mutated isolates, PacC mutants assumed a compact colonial phenotype on supplemented solid DMEM medium pH 7.4 (Figure 1A) which, in contrast to colonies of wild type isolates, lacked peripheral invasive hyphae, and were composed of a denser hyphal network indicative of a hyperbranching morphology (Figure 1B). This compact morphology was pH-independent, being also observed in colonies grown on Aspergillus complete medium pH 6.5 (Figure S2A), where sporulation and pigmentation of ΔpacC mutants was equivalent to that of the wild type. To assess pH sensitivity of the ΔpacC mutants we examined, on pH-buffered minimal media, the extent of radial growth at pHs 8.0 and 7.2, relative to growth at pH 6.5 (Figures S2B and S2C respectively). Consistent with a role for PacC in alkaline adaptation, ΔpacC isolates suffered growth impairment at pH 8.0, achieving approximately 10–20% of the radial growth attained at pH 6.5 (Figure S2B), compared with 40% achieved by wild-type and reconstituted strains. However, sensitivity of ΔpacC isolates to growth at pH 7.2, which approximates the pH of the mammalian lung, did not differ from that of the wild type isolates (Figure S2C).

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