Epithelial uptake leads to fungal killing in vivo and is aberrant in COPD-derived epithelial cells.

Hundreds of spores of Aspergillus fumigatus (Af) are inhaled daily by human beings, representing a constant, possibly fatal, threat to respiratory health. The small size of Af spores suggests that interactions with alveolar epithelial cells (AECs) are frequent; thus, we hypothesized that spore uptake by AECs is important for driving fungal killing and susceptibility to Aspergillus-related disease. Using single-cell approaches to measure spore uptake and its outcomes in vivo, we demonstrate that Af spores are internalized and killed by AECs during whole-animal infection. Moreover, comparative analysis of primary human AECs from healthy and chronic obstructive pulmonary disease (COPD) donors revealed significant alterations in the uptake and killing of spores in COPD-derived AECs. We conclude that AECs contribute to the killing of Af spores and that dysregulation of curative AEC responses in COPD may represent a driver of Aspergillus-related diseases.

TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation.

TIGIT is an inhibitory receptor expressed on lymphocytes and can inhibit T cells by preventing CD226 co-stimulation through interactions in cis or through competition of shared ligands. Whether TIGIT directly delivers cell-intrinsic inhibitory signals in T cells remains unclear. Here we show, by analysing lymphocytes from matched human tumour and peripheral blood samples, that TIGIT and CD226 co-expression is rare on tumour-infiltrating lymphocytes. Using super-resolution microscopy and other techniques, we demonstrate that ligation with CD155 causes TIGIT to reorganise into dense nanoclusters, which coalesce with T cell receptor (TCR)-rich clusters at immune synapses. Functionally, this reduces cytokine secretion in a manner dependent on TIGIT's intracellular ITT-like signalling motif. Thus, we provide evidence that TIGIT directly inhibits lymphocyte activation, acting independently of CD226, requiring intracellular signalling that is proximal to the TCR. Within the subset of tumours where TIGIT-expressing cells do not commonly co-express CD226, this will likely be the dominant mechanism of action.

Aspergillus fumigatus Drives Tissue Damage via Iterative Assaults upon Mucosal Integrity and Immune Homeostasis.

The human lung is constantly exposed to Aspergillus fumigatus spores, the most prevalent worldwide cause of fungal respiratory disease. Pulmonary tissue damage is a unifying feature of Aspergillus-related diseases; however, the mechanistic basis of damage is not understood. In the lungs of susceptible hosts, A. fumigatus undergoes an obligatory morphological switch involving spore germination and hyphal growth. We modeled A. fumigatus infection in cultured A549 human pneumocytes, capturing the phosphoactivation status of five host signaling pathways, nuclear translocation and DNA binding of eight host transcription factors, and expression of nine host response proteins over six time points encompassing exposures to live fungus and the secretome thereof. The resulting data set, comprised of more than 1,000 data points, reveals that pneumocytes mount differential responses to A. fumigatus spores, hyphae, and soluble secreted products via the NF-κB, JNK, and JNK + p38 pathways, respectively. Importantly, via selective degradation of host proinflammatory (IL-6 and IL-8) cytokines and growth factors (FGF-2), fungal secreted products reorchestrate the host response to fungal challenge as well as driving multiparameter epithelial damage, culminating in cytolysis. Dysregulation of NF-κB signaling, involving sequential stimulation of canonical and noncanonical signaling, was identified as a significant feature of host damage both in vitro and in a mouse model of invasive aspergillosis. Our data demonstrate that composite tissue damage results from iterative (repeated) exposures to different fungal morphotypes and secreted products and suggest that modulation of host responses to fungal challenge might represent a unified strategy for therapeutic control of pathologically distinct types of Aspergillus-related disease.

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.

Single-Cell Analysis of Fungal Uptake in Cultured Airway Epithelial Cells Using Differential Fluorescent Staining and Imaging Flow Cytometry.

The respiratory epithelium is the initial point of host contact for inhaled particles, leading to orchestrated, but highly heterogeneous, responses. Human airway epithelial cells (AECs) play a crucial role in host defense by promoting uptake and killing of inhaled microorganisms and concomitant cytokine production in order to recruit professional phagocytes to the site of infection. However, inhaled pathogens can also reside and replicate intracellularly to evade host immune defenses or circulating antimicrobial drugs, ultimately causing apoptosis or cell death of the infected AECs. Imaging flow cytometry (IFC) combines flow cytometry, fluorescent microscopy, and advanced data-processing algorithms to dissect the heterogeneity of the interaction of AECs and inhaled microorganisms and its outcomes at the single-cell level. Here, we describe a novel single-cell approach based on differential fluorescent staining and state-of-the-art IFC to identify, quantify, and analyze individual host-pathogen complexes from cultured AECs infected with spores of the major human fungal pathogen Aspergillus fumigatus.

Automated Quantitative Analysis of Airway Epithelial Cell Detachment Upon Fungal Challenge.

Host-pathogen interactions involve a complex interplay between host and pathogen factors, resulting in either host protective immunity or establishment of disease. One of the hallmarks for disease progression is host tissue destruction. The first host surface to interact with the opportunistic respiratory fungal pathogen, Aspergillus fumigatus, is the airway epithelium. Unravelling the mechanisms involved in airway epithelial cell damage by A. fumigatus is essential to understanding the establishment and progression of infection in the host. Although host cell damage can be measured in vitro by indirect cell lysis assays, here, we describe an automated, simple, and low-cost assay to directly visualize and quantify epithelial cell line damage after challenge with A. fumigatus. We employ the previously characterized tissue noninvasive A. fumigatus ΔpacC mutant to demonstrate the quantitative difference in cell damage relative to its parental tissue invasive strain. This assay is easily scaled up for high-throughput screening of multiple Aspergillus mutants and can be adapted to suit diverse host cell lines, different time points of infection, challenge with other microbes, and drugs or novel compounds.

On the lineage of Aspergillus fumigatus isolates in common laboratory use.

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.

Live-cell imaging of rapid calcium dynamics using fluorescent, genetically-encoded GCaMP probes with Aspergillus fumigatus.

Calcium signalling plays a fundamental role in fungal intracellular signalling. Previous approaches (fluorescent dyes, bioluminescent aequorin, genetically encoded cameleon probes) with imaging rapid subcellular changes in cytosolic free calcium ([Ca2+]c) in fungal cells have produced inconsistent results. Recent data obtained with new fluorescent, genetically encoded GCaMP probes, that are very bright, have resolved this problem. Here, exposing conidia or conidial germlings to high external Ca2+, as an example of an external stressor, induced very dramatic, rapid and dynamic [Ca2+]c changes with localized [Ca2+]c transients and waves. Considerable heterogeneity in the timing of Ca2+ responses of different spores/germlings within the cell population was observed.

Microbial uptake by the respiratory epithelium: outcomes for host and pathogen.

Intracellular occupancy of the respiratory epithelium is a useful pathogenic strategy facilitating microbial replication and evasion of professional phagocytes or circulating antimicrobial drugs. A less appreciated but growing body of evidence indicates that the airway epithelium also plays a crucial role in host defence against inhaled pathogens, by promoting ingestion and quelling of microorganisms, processes that become subverted to favour pathogen activities and promote respiratory disease. To achieve a deeper understanding of beneficial and deleterious activities of respiratory epithelia during antimicrobial defence, we have comprehensively surveyed all current knowledge on airway epithelial uptake of bacterial and fungal pathogens. We find that microbial uptake by airway epithelial cells (AECs) is a common feature of respiratory host-microbe interactions whose stepwise execution, and impacts upon the host, vary by pathogen. Amidst the diversity of underlying mechanisms and disease outcomes, we identify four key infection scenarios and use best-characterised host-pathogen interactions as prototypical examples of each. The emergent view is one in which effi-ciency of AEC-mediated pathogen clearance correlates directly with severity of disease outcome, therefore highlighting an important unmet need to broaden our understanding of the antimicrobial properties of respiratory epithelia and associated drivers of pathogen entry and intracellular fate.

Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance in Aspergillus fumigatus.

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

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.

Mutual independence of alkaline- and calcium-mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus.

Functional coupling of calcium- and alkaline responsive signalling occurs in multiple fungi to afford efficient cation homeostasis. Host microenvironments exert alkaline stress and potentially toxic concentrations of Ca2+ , such that highly conserved regulators of both calcium- (Crz) and pH- (PacC/Rim101) responsive signalling are crucial for fungal pathogenicity. Drugs targeting calcineurin are potent antifungal agents but also perturb human immunity thereby negating their use as anti-infectives, abrogation of alkaline signalling has, therefore, been postulated as an adjunctive antifungal strategy. We examined the interdependency of pH- and calcium-mediated signalling in Aspergillus fumigatus and found that calcium chelation severely impedes hyphal growth indicating a critical requirement for this ion independently of ambient pH. Transcriptomic responses to alkaline pH or calcium excess exhibited minimal similarity. Mutants lacking calcineurin, or its client CrzA, displayed normal alkaline tolerance and nuclear translocation of CrzA was unaffected by ambient pH. Expression of a highly conserved, alkaline-regulated, sodium ATPase was tolerant of genetic or chemical perturbations of calcium-mediated signalling, but abolished in null mutants of the pH-responsive transcription factor PacC, and PacC proteolytic processing occurred normally during calcium excess. Taken together our data demonstrate that in A. fumigatus the regulatory hierarchy governing alkaline tolerance circumvents calcineurin signalling.

Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus.

Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses.

Refining the pH response in Aspergillus nidulans: a modulatory triad involving PacX, a novel zinc binuclear cluster protein.

The Aspergillus nidulans PacC transcription factor mediates gene regulation in response to alkaline ambient pH which, signalled by the Pal pathway, results in the processing of PacC(72) to PacC(27) via PacC(53). 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 palF, specifying the Pal pathway arrestin, probably by PacC(27) and/or PacC(53), prevents an escalating alkaline pH response. Transcript analyses using a reporter and constitutively expressed pacC trans-alleles show that pacC preferential alkaline-expression results from derepression by depletion of the acid-prevalent PacC(72) form. We additionally show that pacC repression requires PacX. pacX mutations suppress PacC processing recalcitrant mutations, in part, through derepressed PacC levels resulting in traces of PacC(27) formed by pH-independent proteolysis. pacX 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.

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

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 ß-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.