Sensory perception in model and pathogenic fungi: Engineering misappropriation of response

The research described characterises pH signalling in the human pathogen Aspergillus fumigatus. Normal functioning of this regulatory system in Aspergillus species requires the integrity of seven genes, including the DNA binding transcription factor PacC, which responds to an alkaline pH signal transmitted by PalA, B, C, F, H and I. Two protein complexes are proposed to sense and transmit the pH signal, the first of which is plasma membrane-localised and composed of two putative pH signal receptors, PalH and PalI, plus a cytoplasmic non-metazoan member of the arrestin family, PalF. Using A. fumigatus transcriptional profiling, Saccharomyces cerevisiae phenotypic screening and the split-ubiquitin MYTH technology, A. fumigatus functions, likely to moderate, or be moderated by the PacC transcription factor, were sought. Two independent A. fumigatus ΔpacC strains have been phenotypically characterised and assessed for virulence in a neutropenic murine model of pulmonary aspergillosis. Of the two, one mutant was further characterised using transcriptional profiling in order to elucidate functions under PacC control during murine infection. The results of this analysis underline the physiological importance of alkaline adaptation during initiation of infection, especially with respect to impacts upon cellular homeostasis, transport and nutrient availability. A S. cerevisiae membrane two-hybrid system has been developed to investigate A. fumigatus plasma membrane protein-protein interactions. Applying the technology, the network of interactions between the components of the A. fumigatus plasma membrane pH signalling complex was explored, and the interaction between the pH-sensing PalH protein and the cytoplasmic arrestin PalF and likely the homodimerisation of PalH were identified. A screen for novel interactors of PalH was also performed, providing 5 candidates for further validation. The S. cerevisiae Δrim101 null mutant and another 80 cation-sensitive null mutants were analysed. An epistacy screen to address the impact of activated Rim101p upon cation sensitivity was performed supporting the identification of putative novel Rim101p/PacC regulators. In parallel the activity of a synthetic Rim101p-regulatable promoter was examined identifying 10 mutants suffering aberrant Rim101p processing and/or transcriptional activity. Finally two A. fumigatus strains have been constructed to functionally express the firefly luciferase gene. Preliminary tests indicate this as a powerful resource to follow murine infection in real time with bioluminescence assays

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Raw data have been deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE54810. Funding: This work was supported in part by grants to EMB from the MRC (G0501164) and BBSRC (BB/G009619/1), to EMB and NDR from the Wellcome Trust (WT093596MA), to MB from Imperial College London (Division of Investigative Sciences PhD Studentship), to HH from the ERA-NET PathoGenoMics project TRANSPAT, Austrian Science Foundation (FWF I282-B09), to SGF from the National Institutes of Health, USA (R01AI073829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Anti-Aspergillus Activities of the Respiratory Epithelium in Health and Disease

Respiratory epithelia fulfil multiple roles beyond that of gaseous exchange, also acting as primary custodians of lung sterility and inflammatory homeostasis. Inhaled fungal spores pose a continual antigenic, and potentially pathogenic, challenge to lung integrity against which the human respiratory mucosa has developed various tolerance and defence strategies. However, respiratory disease and immune dysfunction frequently render the human lung susceptible to fungal diseases, the most common of which are the aspergilloses, a group of syndromes caused by inhaled spores of Aspergillus fumigatus. Inhaled Aspergillus spores enter into a multiplicity of interactions with respiratory epithelia, the mechanistic bases of which are only just becoming recognized as important drivers of disease, as well as possible therapeutic targets. In this mini-review we examine current understanding of Aspergillus-epithelial interactions and, based upon the very latest developments in the field, we explore two apparently opposing schools of thought which view epithelial uptake of Aspergillus spores as either a curative or disease-exacerbating event

The pH-Responsive PacC transcription factor of aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary Aspergillosis

20 p.-8 fig. Margherita Bertuzzi et alt.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 b-glucan receptor. DpacC 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 DpacC 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 DpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.This work was supported in part by grants to EMB from the MRC (G0501164) and BBSRC (BB/G009619/1), to EMB and NDR from the Wellcome Trust(WT093596MA), to MB from Imperial College London (Division of Investigative Sciences PhD Studentship), to HH from the ERA-NET PathoGenoMics project TRANSPAT, Austrian Science Foundation (FWF I282-B09), to SGF from the National Institutes of Health, USA (R01AI073829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Sensory perception in model and pathogenic fungi : engineering misappropriation of response

The research described characterises pH signalling in the human pathogen Aspergillus fumigatus. Normal functioning of this regulatory system in Aspergillus species requires the integrity of seven genes, including the DNA binding transcription factor PacC, which responds to an alkaline pH signal transmitted by PalA, B, C, F, H and I. Two protein complexes are proposed to sense and transmit the pH signal, the first of which is plasma membrane-localised and composed of two putative pH signal receptors, PalH and PalI, plus a cytoplasmic non-metazoan member of the arrestin family, PalF. Using A. fumigatus transcriptional profiling, Saccharomyces cerevisiae phenotypic screening and the split-ubiquitin MYTH technology, A. fumigatus functions, likely to moderate, or be moderated by the PacC transcription factor, were sought. Two independent A. fumigatus ΔpacC strains have been phenotypically characterised and assessed for virulence in a neutropenic murine model of pulmonary aspergillosis. Of the two, one mutant was further characterised using transcriptional profiling in order to elucidate functions under PacC control during murine infection. The results of this analysis underline the physiological importance of alkaline adaptation during initiation of infection, especially with respect to impacts upon cellular homeostasis, transport and nutrient availability. A S. cerevisiae membrane two-hybrid system has been developed to investigate A. fumigatus plasma membrane protein-protein interactions. Applying the technology, the network of interactions between the components of the A. fumigatus plasma membrane pH signalling complex was explored, and the interaction between the pH-sensing PalH protein and the cytoplasmic arrestin PalF and likely the homodimerisation of PalH were identified. A screen for novel interactors of PalH was also performed, providing 5 candidates for further validation. The S. cerevisiae Δrim101 null mutant and another 80 cation-sensitive null mutants were analysed. An epistacy screen to address the impact of activated Rim101p upon cation sensitivity was performed supporting the identification of putative novel Rim101p/PacC regulators. In parallel the activity of a synthetic Rim101p-regulatable promoter was examined identifying 10 mutants suffering aberrant Rim101p processing and/or transcriptional activity. Finally two A. fumigatus strains have been constructed to functionally express the firefly luciferase gene. Preliminary tests indicate this as a powerful resource to follow murine infection in real time with bioluminescence assays.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Novel Insights into Aspergillus fumigatus Pathogenesis and Host Response from State-of-the-Art Imaging of Host–Pathogen Interactions during Infection

Aspergillus fumigatus spores initiate more than 3,000,000 chronic and 300,000 invasive diseases annually, worldwide. Depending on the immune status of the host, inhalation of these spores can lead to a broad spectrum of disease, including invasive aspergillosis, which carries a 50% mortality rate overall; however, this mortality rate increases substantially if the infection is caused by azole-resistant strains or diagnosis is delayed or missed. Increasing resistance to existing antifungal treatments is becoming a major concern; for example, resistance to azoles (the first-line available oral drug against Aspergillus species) has risen by 40% since 2006. Despite high morbidity and mortality, the lack of an in-depth understanding of A. fumigatus pathogenesis and host response has hampered the development of novel therapeutic strategies for the clinical management of fungal infections. Recent advances in sample preparation, infection models and imaging techniques applied in vivo have addressed important gaps in fungal research, whilst questioning existing paradigms. This review highlights the successes and further potential of these recent technologies in understanding the host–pathogen interactions that lead to aspergillosis