title: The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.
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.
authors.affiliation: Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Margherita
authors.initials: M
authors.lastname: Bertuzzi
authors.affiliation: Division of Molecular Biology/Biocenter, Medical University Innsbruck, Innsbruck, Austria; Sandoz GmbH, Kundl, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Schrettl
authors.affiliation: National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Alcazar-Fuoli
authors.affiliation: College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Cairns
authors.affiliation: Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Alberto
authors.initials: A
authors.lastname: Muñoz
authors.affiliation: School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Louise A
authors.initials: LA
authors.lastname: Walker
authors.affiliation: Centre for Molecular Bacteriology & Infection, Imperial College London, London, United Kingdom.
authors.forename: Susanne
authors.initials: S
authors.lastname: Herbst
authors.affiliation: Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Maryam
authors.initials: M
authors.lastname: Safari
authors.affiliation: Centre for Molecular Bacteriology & Infection, Imperial College London, London, United Kingdom.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Cheverton
authors.affiliation: Lab of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland, United States of America.
authors.forename: Dan
authors.initials: D
authors.lastname: Chen
authors.affiliation: David Geffen School of Medicine at University of California, Los Angeles, Los Angeles Biomedical Research Institute at Harbor, University of California, Los Angeles Medical Center, Los Angeles, California, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: Medical Mycology Research Center, Chiba University, Chiba, Japan.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Saijo
authors.affiliation: Lab of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland, United States of America.
authors.forename: Natalie D
authors.initials: ND
authors.lastname: Fedorova
authors.affiliation: Centre for Molecular Bacteriology & Infection, Imperial College London, London, United Kingdom.
authors.forename: Darius
authors.initials: D
authors.lastname: Armstrong-James
authors.affiliation: School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Munro
authors.affiliation: Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Nick D
authors.initials: ND
authors.lastname: Read
authors.affiliation: David Geffen School of Medicine at University of California, Los Angeles, Los Angeles Biomedical Research Institute at Harbor, University of California, Los Angeles Medical Center, Los Angeles, California, United States of America.
authors.forename: Scott G
authors.initials: SG
authors.lastname: Filler
authors.affiliation: Department of Molecular and Cellular Biology, Centro de Investigaciones Biológicas (C.S.I.C.), Madrid, Spain.
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Espeso
authors.affiliation: Lab of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland, United States of America.
authors.forename: William C
authors.initials: WC
authors.lastname: Nierman
authors.affiliation: Division of Molecular Biology/Biocenter, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Hubertus
authors.initials: H
authors.lastname: Haas
authors.affiliation: Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Bignell
mesh_terms: D000818:Animals; D001232:Aspergillus fumigatus / Q000378:metabolism*; D004847:Epithelial Cells / Q000382:microbiology*; D005656:Fungal Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D055732:Pulmonary Aspergillosis / Q000382:microbiology*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; C092394:PacC protein, Aspergillus; D014157:Transcription Factors