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.
Highlights
Spores of the mould pathogen Aspergillus fumigatus are agents of multiple human diseases, most of which initiate with inhalation of fungal spores and, dependent upon host immune status, compromise pulmonary integrity
The pH-responsive A. fumigatus transcription factor PacC is required for epithelial invasion and pathogenicity in leukopenic mice
Consistent with a role for PacC in alkaline adaptation, DpacC 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
Summary
Spores of the mould pathogen Aspergillus fumigatus are agents of multiple human diseases, most of which initiate with inhalation of fungal spores and, dependent upon host immune status, compromise pulmonary integrity. Inhaled spores of the pathogenic mould Aspergillus fumigatus cause fungal lung infections in humans having immune defects. Hyphae degrade the surrounding pulmonary tissue, a process thought to be caused by secreted fungal enzymes; A. fumigatus mutants lacking one or more protease activities retain fully invasive phenotypes in mouse models of disease. Using global transcriptional profiling of wild type and mutant isolates, and in vitro pulmonary invasion assays, we established that loss of PacC leads to a compound non-invasive phenotype characterised by deficits in both contact-mediated epithelial entry and protease expression. Our study sets a new precedent for involvement of both host and pathogen activities in promoting epithelial invasion by A. fumigatus and supports a model wherein fungal protease activity acting subsequently to, or in parallel with, hostmediated epithelial entry provides the mechanistic basis for tissue invasion
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