Epithelial invasion of Aspergillus fumigatus is genetically regulated and requires the transcription factor PACC

Ref ID: 19590

Author:

M Bertuzzi1*, M Schrettl2, L Alcazar-Fuoli3, TC Cairns4, L Walker5, S Herbst6, A Cheverton6,
J Kalchschmidt6, D Chen7, H Liu8, ND Fedorova7, D Armstrong-James6, C Munro5, SG Filler8,
H Haas2, WC Nierman7, EM Bignell1

Author address:

1Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester,
Manchester, UK
2Dept. of Molecular Biology, Medical University Innsbrück, Innsbrück, Austria
3Mycology Reference laboratory, National Centre for Mi

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
A hallmark of invasive lung diseases caused by Aspergillus fumigatus is the penetrative growth
of fungal hyphae which destroy pulmonary tissue. Although the respiratory epithelium represents
the first point of contact with the host, the molecular basis of the host-pathogen interaction at this
interface remains, for aspergilloses, poorly characterised. We established that the transcription factor
PacC is required for pulmonary invasion as 916;pacC mutants are non-invasive, and attenuated for
virulence, in a leukopenic model of infection. The purpose of this study was to interrogate the
molecular basis of this critical deficit.
Methods:
To assess the effect of A. fumigatus spores on epithelial cells (ECs), wild type and 916;pacC strains
were co-incubated with A549 monolayers and the number of detaching cells was enumerated at 16
hours of co-incubation. The assay was repeated using fungal culture filtrates, cell wall extracts, and
heat killed hyphae to determine the role, respectively, of secreted proteins, cell wall components and
physical perturbation in monolayer decay. To identify gene functions under PacC regulatory control
during murine infection we performed transcriptional profiling of the A. fumigatus ATCC46645
clinical isolate and an isogenic 916;pacC mutant using germlings extracted from bronchoalveolar
lavages (BALs) of leukopenic mice, after 4, 8, 12 and 16 hours of infection. To determine the effect
of spore internalisation upon epithelial integrity a modified nystatin assay was used to measure
the number of wild type and 916;pacC spores internalised during the first 4 hours of host-pathogen
contact. This assay was also performed in the presence of Cytochalasin D and a Dectin-1 blocking
antibody to study the contribution of actin and Dectin-1, respectively, to the internalisation process.
Susceptibility of the isolates to caspofungin was measured in vitro according to the EUCAST
specifications, and during murine infection by A. fumigatus viable counts and histological analysis
of lung sections.
Results:
Using our in vitro infection assays, we found that A. fumigatus-mediated decay of A549 monolayers
occurs via two temporally and mechanistically distinct processes, involving an initial contactdependent
mechanism, followed by damage caused by soluble effectors. Clues to the molecular
basis of these perturbations were provided by transcriptional profiling of the 916;pacC mutant during
murine infection which revealed aberrant expression of secreted proteases and remodelling of the
fungal cell wall during infectious growth. Cell wall components and internalisation of spores by ECs
contribute to epithelial decay during the early phase of the host-pathogen interaction. PacC mutants
are deficient in both of these processes and nystatin protection assays revealed that 916;pacC spores
are internalised less efficiently by ECs relative to the respective parental isolates. Importantly, pH
non-sensing mutants were demonstrated to be more susceptible to caspofungin treatment, both in
vitro and during murine infection.
Conclusions:
Epithelial invasion is a genetically regulated trait, and is regulated by the PacC transcription factor,
which mediates distinct phases of host perturbation during pulmonary aspergillosis. In light of the
hypersensitivity of infecting 916;pacC mutants to cell wall-active antifungal agents, our study identifies
PacC signalling as a premier target for antifungal therapy.

Abstract Number: 115

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

New link: NULL

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