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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 hypersensitive to cell wall active drugs.(A)In vitro susceptibility testing to caspofungin (CFN). Determination of CFN MEC (μg/ml), twice in triplicates, 1.25 × 105 spores, 48 hr. (B)A. fumigatus growth in the presence of 16 µg/ml CFN. 400× magnification. (C) Histopathology from leukopenic mice infected with CEA10 or ΔpacCCEA10 (3 × 104 spores) in the presence or absence of 5 mg/kg CFN. Lungs sections at 24 hr of infection, GMS and light green staining, 200× magnification. (D)In vivo susceptibility testing to caspofungin. Fungal burden at 48 hr of infection, calculated by CFUs, from leukopenic mice infected as from C. Results are expressed as CFUs per gram of lung tissue. A and D: unpaired t test, *** p<0.001, **.001<p<0.01, and * 0.01<p<0.05.
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ppat-1004413-g008: ΔpacC mutants are hypersensitive to cell wall active drugs.(A)In vitro susceptibility testing to caspofungin (CFN). Determination of CFN MEC (μg/ml), twice in triplicates, 1.25 × 105 spores, 48 hr. (B)A. fumigatus growth in the presence of 16 µg/ml CFN. 400× magnification. (C) Histopathology from leukopenic mice infected with CEA10 or ΔpacCCEA10 (3 × 104 spores) in the presence or absence of 5 mg/kg CFN. Lungs sections at 24 hr of infection, GMS and light green staining, 200× magnification. (D)In vivo susceptibility testing to caspofungin. Fungal burden at 48 hr of infection, calculated by CFUs, from leukopenic mice infected as from C. Results are expressed as CFUs per gram of lung tissue. A and D: unpaired t test, *** p<0.001, **.001<p<0.01, and * 0.01<p<0.05.

Mentions: The fungal cell wall is a premier, pathogen-specific target for antifungal drugs. Given the significant cell wall defect observed in ΔpacC mutants we predicted altered echinocandin sensitivity relative to wild type isolates. A standard EUCAST assay [53] was used to calculate the susceptibility of isolates, revealing increased susceptibility (Figure 8A) of ΔpacC mutants (minimum effective concentration, MEC, of 0.11 µg/ml) compared to that of ATCC46645 (∼ 0.58 µg/ml) and CEA10 (∼ 0.75 µg/ml). A. fumigatus strains grown in the presence of 16 µg/ml caspofungin displayed aberrant morphology, elevated branching and shortening of hyphae. Heightened severity of these phenotypes was observed for ΔpacC mutants which demonstrated extensive ballooning of hyphal tips (Figure 8B). Given the tendency for chitin increase to promote echinocandin tolerance the heightened susceptibility of ΔpacC mutants is surprising; however, an obvious explanation for this effect would be increased porosity due to altered cell wall architecture.


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 hypersensitive to cell wall active drugs.(A)In vitro susceptibility testing to caspofungin (CFN). Determination of CFN MEC (μg/ml), twice in triplicates, 1.25 × 105 spores, 48 hr. (B)A. fumigatus growth in the presence of 16 µg/ml CFN. 400× magnification. (C) Histopathology from leukopenic mice infected with CEA10 or ΔpacCCEA10 (3 × 104 spores) in the presence or absence of 5 mg/kg CFN. Lungs sections at 24 hr of infection, GMS and light green staining, 200× magnification. (D)In vivo susceptibility testing to caspofungin. Fungal burden at 48 hr of infection, calculated by CFUs, from leukopenic mice infected as from C. Results are expressed as CFUs per gram of lung tissue. A and D: unpaired t test, *** p<0.001, **.001<p<0.01, and * 0.01<p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004413-g008: ΔpacC mutants are hypersensitive to cell wall active drugs.(A)In vitro susceptibility testing to caspofungin (CFN). Determination of CFN MEC (μg/ml), twice in triplicates, 1.25 × 105 spores, 48 hr. (B)A. fumigatus growth in the presence of 16 µg/ml CFN. 400× magnification. (C) Histopathology from leukopenic mice infected with CEA10 or ΔpacCCEA10 (3 × 104 spores) in the presence or absence of 5 mg/kg CFN. Lungs sections at 24 hr of infection, GMS and light green staining, 200× magnification. (D)In vivo susceptibility testing to caspofungin. Fungal burden at 48 hr of infection, calculated by CFUs, from leukopenic mice infected as from C. Results are expressed as CFUs per gram of lung tissue. A and D: unpaired t test, *** p<0.001, **.001<p<0.01, and * 0.01<p<0.05.
Mentions: The fungal cell wall is a premier, pathogen-specific target for antifungal drugs. Given the significant cell wall defect observed in ΔpacC mutants we predicted altered echinocandin sensitivity relative to wild type isolates. A standard EUCAST assay [53] was used to calculate the susceptibility of isolates, revealing increased susceptibility (Figure 8A) of ΔpacC mutants (minimum effective concentration, MEC, of 0.11 µg/ml) compared to that of ATCC46645 (∼ 0.58 µg/ml) and CEA10 (∼ 0.75 µg/ml). A. fumigatus strains grown in the presence of 16 µg/ml caspofungin displayed aberrant morphology, elevated branching and shortening of hyphae. Heightened severity of these phenotypes was observed for ΔpacC mutants which demonstrated extensive ballooning of hyphal tips (Figure 8B). Given the tendency for chitin increase to promote echinocandin tolerance the heightened susceptibility of ΔpacC mutants is surprising; however, an obvious explanation for this effect would be increased porosity due to altered cell wall architecture.

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