Inferring fungal growth rates from optical density data.

Quantifying fungal growth underpins our ability to effectively treat severe fungal infections. Current methods quantify fungal growth rates from time-course morphology-specific data, such as hyphal length data. However, automated large-scale collection of such data lies beyond the scope of most clinical microbiology laboratories. In this paper, we propose a mathematical model of fungal growth to estimate morphology-specific growth rates from easy-to-collect, but indirect, optical density (OD600) data of Aspergillus fumigatus growth (filamentous fungus). Our method accounts for OD600 being an indirect measure by explicitly including the relationship between the indirect OD600 measurements and the calibrating true fungal growth in the model. Therefore, the method does not require de novo generation of calibration data. Our model outperformed reference models at fitting to and predicting OD600 growth curves and overcame observed discrepancies between morphology-specific rates inferred from OD600 versus directly measured data in reference models that did not include calibration.

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response.

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed "aspergilloses," in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host-pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients' lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host-pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

Pathogenesis of Respiratory Viral and Fungal Coinfections.

Individuals suffering from severe viral respiratory tract infections have recently emerged as "at risk" groups for developing invasive fungal infections. Influenza virus is one of the most common causes of acute lower respiratory tract infections worldwide. Fungal infections complicating influenza pneumonia are associated with increased disease severity and mortality, with invasive pulmonary aspergillosis being the most common manifestation. Strikingly, similar observations have been made during the current coronavirus disease 2019 (COVID-19) pandemic. The copathogenesis of respiratory viral and fungal coinfections is complex and involves a dynamic interplay between the host immune defenses and the virulence of the microbes involved that often results in failure to return to homeostasis. In this review, we discuss the main mechanisms underlying susceptibility to invasive fungal disease following respiratory viral infections. A comprehensive understanding of these interactions will aid the development of therapeutic modalities against newly identified targets to prevent and treat these emerging coinfections.

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.

Heightened Efficacy of Anidulafungin When Used in Combination with Manogepix or 5-Flucytosine against Candida auris In Vitro.

Candida auris is an emerging, multidrug-resistant fungal pathogen that causes refractory colonization and life-threatening, invasive nosocomial infections. The high proportion of C. auris isolates that display antifungal resistance severely limits treatment options. Combination therapies provide a possible strategy by which to enhance antifungal efficacy and prevent the emergence of further resistance. Therefore, we examined drug combinations using antifungals that are already in clinical use or are undergoing clinical trials. Using checkerboard assays, we screened combinations of 5-flucytosine and manogepix (the active form of the novel antifungal drug fosmanogepix) with anidulafungin, amphotericin B, or voriconazole against drug resistant and susceptible C. auris isolates from clades I and III. Fractional inhibitory concentration indices (FICI values) of 0.28 to 0.75 and 0.36 to 1.02 were observed for combinations of anidulafungin with manogepix or 5-flucytosine, respectively, indicating synergistic activity. The high potency of these anidulafungin combinations was confirmed using live-cell microfluidics-assisted imaging of the fungal growth. In summary, combinations of anidulafungin with manogepix or 5-flucytosine show great potential against both resistant and susceptible C. auris isolates.

Exploring a novel genomic safe-haven site in the human pathogenic mould Aspergillus fumigatus.

Aspergillus fumigatus is the most important airborne fungal pathogen and allergen of humans causing high morbidity and mortality worldwide. The factors that govern pathogenicity of this organism are multi-factorial and are poorly understood. Molecular tools to dissect the mechanisms of pathogenicity in A. fumigatus have improved significantly over the last 20 years however many procedures have not been standardised for A. fumigatus. Here, we present a new genomic safe-haven locus at the site of an inactivated transposon, named SH-aft4, which can be used to insert DNA sequences in the genome of this fungus without impacting its phenotype. We show that we are able to effectively express a transgene construct from the SH-aft4 and that natural regulation of promoter function is conserved at this site. Furthermore, the SH-aft4 locus is highly conserved in the genome of a wide range of clinical and environmental isolates including the isolates commonly used by many laboratories CEA10, Af293 and ATCC46645, allowing a wide range of isolates to be manipulated. Our results show that the aft4 locus can serve as a site for integration of a wide range of genetic constructs to aid functional genomics studies of this important human fungal pathogen.

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.

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.

Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus.

Aspergillus fumigatus is a saprophytic fungal pathogen that is the cause of more than 300,000 life-threatening infections annually. Our understanding of pathogenesis and factors contributing to disease progression are limited. Development of rapid and versatile gene editing methodologies for A. fumigatus is essential. CRISPR-Cas9 mediated transformation has been widely used as a novel genome editing tool and has been used for a variety of editing techniques, such as protein tagging, gene deletions and site-directed mutagenesis in A. fumigatus. However, successful genome editing relies on time consuming, multi-step cloning procedures paired with the use of selection markers, which can result in a metabolic burden for the host and/or unintended transcriptional modifications at the site of integration. We have used an in vitro CRISPR-Cas9 assembly methodology to perform selection-free genome editing, including epitope tagging of proteins and site-directed mutagenesis. The repair template used during this transformation use 50 bp micro-homology arms and can be generated with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.

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.

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.

Transcript levels of the Aspergillus fumigatus Cdc42 module, polarisome, and septin genes show little change from dormancy to polarity establishment.

Aspergillus fumigatus is the most common airborne pathogen causing fatal mycoses in immunocompromised patients. During the first 8 hours of development A. fumigatus conidia break dormancy, expand isotopically, establish an axis of polarity, and begin to extend germ tubes in a polar manner. The transition from isotropic to polar growth is critical for tissue invasion and pathogenesis. In the current work, we used two-color microarrays to examine the A. fumigatus transcriptome during early development, focusing on the isotropic to polar switch. The most highly regulated transcripts in the isotropic to polar switch did not include known polarity genes. Transcripts encoding the Cdc42 module, polarisome components, and septins, known to be critical players in polarity, showed relatively steady levels during the isotropic to polar switch. Indeed, these transcripts were present in dormant conidia, and their levels changed little from dormancy through germ tube emergence. Not only did the isotropic to polar switch show little change in the expression of key polarity genes of the Cdc42 module, polarisome, and septins, it also showed the lowest overall levels of both up- and downregulation in early development.

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.

Mevalonate governs interdependency of ergosterol and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the most common airborne fungal pathogen for humans. In this mold, iron starvation induces production of the siderophore triacetylfusarinine C (TAFC). Here we demonstrate a link between TAFC and ergosterol biosynthetic pathways, which are both critical for virulence and treatment of fungal infections. Consistent with mevalonate being a limiting prerequisite for TAFC biosynthesis, we observed increased expression of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (Hmg1) under iron starvation, reduced TAFC biosynthesis following lovastatin-mediated Hmg1 inhibition, and increased TAFC biosynthesis following Hmg1 overexpression. We identified enzymes, the acyl-CoA ligase SidI and the enoyl-CoA hydratase SidH, linking biosynthesis of mevalonate and TAFC, deficiency of which under iron starvation impaired TAFC biosynthesis, growth, oxidative stress resistance, and murine virulence. Moreover, inactivation of these enzymes alleviated TAFC-derived biosynthetic demand for mevalonate, as evidenced by increased resistance to lovastatin. Concordant with bilateral demand for mevalonate, iron starvation decreased the ergosterol content and composition, a phenotype that is mitigated in TAFC-lacking mutants.

Distribution, expression and expansion of Aspergillus fumigatus LINE-like retrotransposon populations in clinical and environmental isolates.

Functional genomic analysis of the mould pathogen Aspergillus fumigatus has identified multiple secondary metabolism genes upregulated in the host niche. Intriguingly, transcriptomic analyses of infectious germlings, germinating spores and mutants lacking the LaeA methyltransferase reveal differential expression of transposable elements (TEs), which often flank secondary metabolite gene clusters. In this study we investigate, in clinical and environmental isolates, the structure and distribution of a specific class of A. fumigatus long interspersed nuclear element (LINE)-like retrotransposons occupying subtelomeric loci in the A. fumigatus genome, and probe their stability in response to laboratory- and host-imposed stresses. In silico analyses revealed that this class belongs to the Tad clade of LINE-like elements. Southern blotting with a LINE-specific probe in clinical and environmental isolates revealed a high variability in the insertion pattern between strains and active transcription of LINE-like element(s) was discernable, in the type strain Af293, by RT-PCR. One out of 14 tested clinical isolates did not contain any LINEs at all, arguing against an absolute requirement for LINE-mediated activities in human infections. Finally, we found preliminary evidence of an association between mycovirus-infection and the expansion of Tad-element populations in discrete A. fumigatus genomes.

Clonality despite sex: the evolution of host-associated sexual neighborhoods in the pathogenic fungus Penicillium marneffei.

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.

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.

Functional analysis of the Aspergillus fumigatus kinome identifies a druggable DYRK kinase that regulates septal plugging.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

The pathobiology of human fungal infections.

Human fungal infections are a historically neglected area of disease research, yet they cause more than 1.5 million deaths every year. Our understanding of the pathophysiology of these infections has increased considerably over the past decade, through major insights into both the host and pathogen factors that contribute to the phenotype and severity of these diseases. Recent studies are revealing multiple mechanisms by which fungi modify and manipulate the host, escape immune surveillance and generate complex comorbidities. Although the emergence of fungal strains that are less susceptible to antifungal drugs or that rapidly evolve drug resistance is posing new threats, greater understanding of immune mechanisms and host susceptibility factors is beginning to offer novel immunotherapeutic options for the future. In this Review, we provide a broad and comprehensive overview of the pathobiology of human fungal infections, focusing specifically on pathogens that can cause invasive life-threatening infections, highlighting recent discoveries from the pathogen, host and clinical perspectives. We conclude by discussing key future challenges including antifungal drug resistance, the emergence of new pathogens and new developments in modern medicine that are promoting susceptibility to infection.