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.

The origin of isolates routinely used by the community of Aspergillus fumigatus researchers is periodically a matter of intense discussion at our centre, as the construction of recombinant isolates have sometimes followed convoluted routes, the documentation describing their lineages is fragmented, and the nomenclature is confusing. As an aide memoir, not least for our own benefit, we submit the following account and tabulated list of strains (Table 1) in an effort to collate all of the relevant information in a single, easily accessible document. To maximise the accuracy of this record we have consulted widely amongst the community of Medical Mycologists using these strains. All the strains described are currently available from one of these organisations, namely the Fungal Genetics Stock Centre (FGSC), FungiDB, Ensembl Fungi and The National Collection of Pathogenic Fungi (NCPF) at Public Health England. Display items from this manuscript are also featured on FungiDB. Lay abstract We present a concise overview on the definition, origin and unique genetic makeup of the Aspergillus fumigatus isolates routinely in use by the fungal research community, to aid researchers to describe past and new strains and the experimental differences observed more accurately.

Abstract: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.

29The antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is 30 licenced for treatment of fungal diseases however it is rarely used as a monotherapeutic 31 to treat Aspergillus infection. Despite being potent against other fungal pathogens, 5FC 32 has limited activity against A. fumigatus when standard in vitro assays are used to 33 determine susceptibility. However, in modified in vitro assays where the pH is set to pH 34 5 the activity of 5FC increases significantly. 35Here we provide evidence that fcyB, a gene that encodes a purine-cytosine permease 36 orthologous to known 5FC importers is downregulated at pH 7 and is the primary factor 37 responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional 38 regulators that are responsible for repression of fcyB and consequently mediators of 5FC 39 resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We 40propose that the activity of 5FC might be enhanced by perturbation of factors that 41repress fcyB expression such as PacC or other components of the pH sensing machinery.

SummaryThe A spergillus nidulans PacC transcription factor mediates gene regulation in response to alkaline ambient pH which, signalled by the Pal pathway, results in the processing of PacC72 to PacC27 via PacC53. Here we investigate two levels at which the pH regulatory system is transcriptionally moderated by pH and identify and characterise a new component of the pH regulatory machinery, PacX. Transcript level analysis and overexpression studies demonstrate that repression of acid‐expressed pal F, specifying the Pal pathway arrestin, probably by PacC27 and/or PacC53, prevents an escalating alkaline pH response. Transcript analyses using a reporter and constitutively expressed pac C  trans‐alleles show that pac C preferential alkaline‐expression results from derepression by depletion of the acid‐prevalent PacC72 form. We additionally show that pac C repression requires PacX. pac X mutations suppress PacC processing recalcitrant mutations, in part, through derepressed PacC levels resulting in traces of PacC27 formed by pH‐independent proteolysis. pac X was cloned by impala transposon mutagenesis. PacX, with homologues within the Leotiomyceta, has an unusual structure with an amino‐terminal coiled‐coil and a carboxy‐terminal zinc binuclear cluster. pacX mutations indicate the importance of these regions. One mutation, an unprecedented finding in A . nidulans genetics, resulted from an insertion of an endogenous Fot1‐like transposon.

Aspergillus fumigatus is the most pathogenic species among the Aspergilli, and the major fungal agent of human pulmonary infection. To prosper in diverse ecological niches, Aspergilli have evolved numerous mechanisms for adaptive gene regulation, some of which are also crucial for mammalian infection. Among the molecules which govern such responses, integral membrane receptors are thought to be the most amenable to therapeutic modulation. This is due to the localization of these molecular sensors at the periphery of the fungal cell, and to the prevalence of small molecules and licensed drugs which target receptor-mediated signaling in higher eukaryotic cells. In this review we highlight the progress made in characterizing receptor-mediated environmental adaptation in A. fumigatus and its relevance for pathogenicity in mammals. By presenting a first genomic survey of integral membrane proteins in this organism, we highlight an abundance of putative seven transmembrane domain (7TMD) receptors, the majority of which remain uncharacterized. Given the dependency of A. fumigatus upon stress adaptation for colonization and infection of mammalian hosts, and the merits of targeting receptor-mediated signaling as an antifungal strategy, a closer scrutiny of sensory perception and signal transduction in this organism is warranted.

Hundreds of spores of the common mould Aspergillus fumigatus (Af) are inhaled daily by human beings, representing a constant, often fatal, threat to our respiratory health. The small size of Af spores suggest that interactions with Airway Epithelial Cells (AECs) are frequent and we and others have previously demonstrated that AECs are able to internalise Af spores. We thus hypothesised that Af spore uptake and killing by AECs is important for driving efficient fungal clearance in vivo and that defective spore uptake and killing would represent major risk factors for Aspergillus-related diseases. In order to test this, we utilised single-cell approaches based on Imaging Flow Cytometry (IFC) and live-cell microfluidic imaging to measure spore uptake and outcomes in vitro, in vivo and using primary human AECs. In vitro, viability of immortalised AECs was largely unaffected by Af uptake and AECs were able to significantly curtail the growth of internalised spores. Applying our approach directly to infected mouse lungs we demonstrated, for the first time, that Af spores are internalised and killed by AECs during whole animal infection, whereby only ~3% of internalised spores remained viable after 8 hours of co-incubation with murine AECs. Finally, in vitro analysis of primary human AECs from healthy and at-risk donors revealed significant alterations in the uptake and consequent outcomes in Chronic Obstructive Pulmonary Disease (COPD), whereby gorging COPD-derived AECs were unable to quell intracellular Af as efficiently as healthy primary AECs. We have thus demonstrated that AECs efficiently kill Af spores upon uptake in vivo and that this process is altered in COPD, a well-known risk factor for debilitating fungal lung disease, thereby suggesting that AECs critically contribute to the efficient clearance of inhaled Af spores and that dysregulation of curative AEC responses represents a potent driver of Aspergillus-related diseases.

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