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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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Aberrant morphology of A. fumigatus ΔpacC mutants impacts contact-mediated epithelial decay.(A) Calcofluor white staining of A. fumigatus strains after growth for 17–18 hr in AMM (pH 6.5) at 28°C. (B) Quantification of calcofluor white-mediated fluorescence (arbitrary units) as measured from panel A. (C) Percentage, in cell wall extracts relative to total weight, of chitin. (D) Percentage, in cell wall extracts relative to total weight, of glucan. (E) Percentage, in cell wall extracts relative to total weight, of mannan. (F) Percent detachment, relative to PBS challenge, of A549 cells after co-incubation with cell wall extracts (10 µg/ml). Significance was calculated relative to PBS-challenged monolayers. (G) Percent aberrant morphology, amongst thimerosal-killed fungus-proximal ECs, per unit of hyphal length, relative to wild-type challenged monolayers. B and C: biological and technical triplicates. D and E: performed twice in triplicate. B, C and D: unpaired t test. E: 1-way ANOVA test. *** p<0.001 and ** 0.001<p<0.01.
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ppat-1004413-g005: Aberrant morphology of A. fumigatus ΔpacC mutants impacts contact-mediated epithelial decay.(A) Calcofluor white staining of A. fumigatus strains after growth for 17–18 hr in AMM (pH 6.5) at 28°C. (B) Quantification of calcofluor white-mediated fluorescence (arbitrary units) as measured from panel A. (C) Percentage, in cell wall extracts relative to total weight, of chitin. (D) Percentage, in cell wall extracts relative to total weight, of glucan. (E) Percentage, in cell wall extracts relative to total weight, of mannan. (F) Percent detachment, relative to PBS challenge, of A549 cells after co-incubation with cell wall extracts (10 µg/ml). Significance was calculated relative to PBS-challenged monolayers. (G) Percent aberrant morphology, amongst thimerosal-killed fungus-proximal ECs, per unit of hyphal length, relative to wild-type challenged monolayers. B and C: biological and technical triplicates. D and E: performed twice in triplicate. B, C and D: unpaired t test. E: 1-way ANOVA test. *** p<0.001 and ** 0.001<p<0.01.

Mentions: Amongst the functional cohorts aberrantly regulated during ΔpacCATCC infections (Figures S5–S7 and Dataset S2) cell wall biosynthesis offered a plausible mechanism for contact-dependent host damage. To analyse cell wall compositions of mutant and wild-type isolates, strains were stained with the chitin-binding agent calcofluor white (CFW). Microscopic examination revealed intensified CFW-staining of ΔpacC germ tube tips relative to those of the parental isolates (Figure 5A) and quantitative analysis of fluorescence intensities revealed significantly higher CFW in ΔpacC germ tubes (Figure 5B). In addition, electron microscopy showed a thickened cell wall in the ΔpacCATCC mutant, which was highly evident after 16 hours of growth (Figure S10). Hyphal cell wall composition (Figures 5C, D and E) was assessed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). After 16 hours of growth, cell wall chitin content was found to be 20% higher in extracts from ΔpacC isolates, relative to wild-type cells (Figure 5C). The quantity of cell wall glucan and mannan was measured as equivalent between mutant and wild type cells (Figures 5D and 5E).


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)

Aberrant morphology of A. fumigatus ΔpacC mutants impacts contact-mediated epithelial decay.(A) Calcofluor white staining of A. fumigatus strains after growth for 17–18 hr in AMM (pH 6.5) at 28°C. (B) Quantification of calcofluor white-mediated fluorescence (arbitrary units) as measured from panel A. (C) Percentage, in cell wall extracts relative to total weight, of chitin. (D) Percentage, in cell wall extracts relative to total weight, of glucan. (E) Percentage, in cell wall extracts relative to total weight, of mannan. (F) Percent detachment, relative to PBS challenge, of A549 cells after co-incubation with cell wall extracts (10 µg/ml). Significance was calculated relative to PBS-challenged monolayers. (G) Percent aberrant morphology, amongst thimerosal-killed fungus-proximal ECs, per unit of hyphal length, relative to wild-type challenged monolayers. B and C: biological and technical triplicates. D and E: performed twice in triplicate. B, C and D: unpaired t test. E: 1-way ANOVA test. *** p<0.001 and ** 0.001<p<0.01.
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Related In: Results  -  Collection

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ppat-1004413-g005: Aberrant morphology of A. fumigatus ΔpacC mutants impacts contact-mediated epithelial decay.(A) Calcofluor white staining of A. fumigatus strains after growth for 17–18 hr in AMM (pH 6.5) at 28°C. (B) Quantification of calcofluor white-mediated fluorescence (arbitrary units) as measured from panel A. (C) Percentage, in cell wall extracts relative to total weight, of chitin. (D) Percentage, in cell wall extracts relative to total weight, of glucan. (E) Percentage, in cell wall extracts relative to total weight, of mannan. (F) Percent detachment, relative to PBS challenge, of A549 cells after co-incubation with cell wall extracts (10 µg/ml). Significance was calculated relative to PBS-challenged monolayers. (G) Percent aberrant morphology, amongst thimerosal-killed fungus-proximal ECs, per unit of hyphal length, relative to wild-type challenged monolayers. B and C: biological and technical triplicates. D and E: performed twice in triplicate. B, C and D: unpaired t test. E: 1-way ANOVA test. *** p<0.001 and ** 0.001<p<0.01.
Mentions: Amongst the functional cohorts aberrantly regulated during ΔpacCATCC infections (Figures S5–S7 and Dataset S2) cell wall biosynthesis offered a plausible mechanism for contact-dependent host damage. To analyse cell wall compositions of mutant and wild-type isolates, strains were stained with the chitin-binding agent calcofluor white (CFW). Microscopic examination revealed intensified CFW-staining of ΔpacC germ tube tips relative to those of the parental isolates (Figure 5A) and quantitative analysis of fluorescence intensities revealed significantly higher CFW in ΔpacC germ tubes (Figure 5B). In addition, electron microscopy showed a thickened cell wall in the ΔpacCATCC mutant, which was highly evident after 16 hours of growth (Figure S10). Hyphal cell wall composition (Figures 5C, D and E) was assessed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). After 16 hours of growth, cell wall chitin content was found to be 20% higher in extracts from ΔpacC isolates, relative to wild-type cells (Figure 5C). The quantity of cell wall glucan and mannan was measured as equivalent between mutant and wild type cells (Figures 5D and 5E).

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