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.
To better combat the expansion of antibiotic resistance in pathogens, new compounds, particularly those with novel mechanisms-of-action MOA, represent a major research priority in biomedical science. ...However, rediscovery of known antibiotics demonstrates a need for approaches that accurately identify potential novelty with higher throughput and reduced labor. Here we describe an explainable artificial intelligence classification methodology that emphasizes prediction performance and human interpretability by using a Hierarchical Ensemble of Classifiers model optimized with a novel feature selection algorithm called Clairvoyance; collectively referred to as a CoHEC model. We evaluated our methods using whole transcriptome responses from Escherichia coli challenged with 41 known antibiotics and 9 crude extracts while depositing 122 transcriptomes unique to this study. Our CoHEC model can properly predict the primary MOA of previously unobserved compounds in both purified forms and crude extracts at an accuracy above 99%, while also correctly identifying darobactin, a newly discovered antibiotic, as having a novel MOA. In addition, we deploy our methods on a recent E. coli transcriptomics dataset from a different strain and a Mycobacterium smegmatis metabolomics timeseries dataset showcasing exceptionally high performance; improving upon the performance metrics of the original publications. We not only provide insight into the biological interpretation of our model but also that the concept of MOA is a non-discrete heuristic with diverse effects for different compounds within the same MOA, suggesting substantial antibiotic diversity awaiting discovery within existing MOA.
Fungi produce an impressive array of secondary metabolites (SMs) including mycotoxins, antibiotics and pharmaceuticals. The genes responsible for their biosynthesis, export, and transcriptional ...regulation are often found in contiguous gene clusters. To facilitate annotation of these clusters in sequenced fungal genomes, we developed the web-based software SMURF (
www.jcvi.org/smurf/) to systematically predict clustered SM genes based on their genomic context and domain content. We applied SMURF to catalog putative clusters in 27 publicly available fungal genomes. Comparison with genetically characterized clusters from six fungal species showed that SMURF accurately recovered all clusters and detected additional potential clusters. Subsequent comparative analysis revealed the striking biosynthetic capacity and variability of the fungal SM pathways and the correlation between unicellularity and the absence of SMs. Further genetics studies are needed to experimentally confirm these clusters.
The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen ...Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated.
Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25-36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics.
The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing.
The opportunistic pathogens Burkholderia cepacia and Burkholderia contaminans, both genomovars of the Burkholderia cepacia complex (BCC), are frequently cultured from the potable water dispenser ...(PWD) of the International Space Station (ISS). Here, we sequenced the genomes and conducted phenotypic assays to characterize these Burkholderia isolates. All recovered isolates of the two species fall within monophyletic clades based on phylogenomic trees of conserved single-copy core genes. Within species, the ISS-derived isolates all demonstrate greater than 99% average nucleotide identity (with 95-99% of genomes aligning) and share around 90% of the identified gene clusters from a pangenomic analysis-suggesting that the two groups are each composed of highly similar genomic lineages and their members may have all stemmed from the same two founding populations. The differences that can be observed between the recovered isolates at the pangenomic level are primarily located within putative plasmids. Phenotypically, macrophage intracellularization and lysis occurred at generally similar rates between all ISS-derived isolates, as well as with their respective type-terrestrial strain references. All ISS-derived isolates exhibited antibiotic sensitivity similar to that of the terrestrial reference strains, and minimal differences between isolates were observed. With a few exceptions, biofilm formation rates were generally consistent across each species. And lastly, though isolation date does not necessarily provide any insight into how long a given isolate had been aboard the ISS, none of the assayed physiology correlated with either date of isolation or distances based on nucleotide variation. Overall, we find that while the populations of Burkholderia present in the ISS PWS each maintain virulence, they are likely are not more virulent than those that might be encountered on planet and remain susceptible to clinically used antibiotics.
Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using ...mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.
Summary
Fungi of the genus Aspergillus are widespread in the environment. Some Aspergillus species, most commonly Aspergillus fumigatus, may lead to a variety of allergic reactions and ...life‐threatening systemic infections in humans. Invasive aspergillosis occurs primarily in patients with severe immunodeficiency, and has dramatically increased in recent years. There are several factors at play that contribute to aspergillosis, including both fungus and host‐related factors such as strain virulence and host pulmonary structure/immune status, respectively. The environmental tenacity of Aspergilllus, its dominance in diverse microbial communities/habitats, and its ability to navigate the ecophysiological and biophysical challenges of host infection are attributable, in large part, to a robust stress‐tolerance biology and exceptional capacity to generate cell‐available energy. Aspects of its stress metabolism, ecology, interactions with diverse animal hosts, clinical presentations and treatment regimens have been well‐studied over the past years. Here, we synthesize these findings in relation to the way in which some Aspergillus species have become successful opportunistic pathogens of human‐ and other animal hosts. We focus on the biophysical capabilities of Aspergillus pathogens, key aspects of their ecophysiology and the flexibility to undergo a sexual cycle or form cryptic species. Additionally, recent advances in diagnosis of the disease are discussed as well as implications in relation to questions that have yet to be resolved.
Some Aspergillus species, most commonly Aspergillus fumigatus, can induce allergic reactions and life‐threatening systemic infections in humans. Here, we discuss the potency of Aspergillus as an opportunistic pathogen of humans and other animal hosts. We focus on the biophysical capabilities of pathogenic aspergilli; key aspects of their ecophysiology; and their ability to undergo a sexual cycle or form cryptic species. Recent advances in diagnosis of the disease are discussed, as well as outstanding scientific questions.
Aspergillus flavus is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or ...more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within A. flavus, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 μm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 μm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an A. flavus S morphotype, strain AF70. We provide a comprehensive comparison of the A. flavus S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters.
Aspergillus fumigatus is a versatile fungus able to successfully exploit diverse environments from mammalian lungs to agricultural waste products. Among its many fitness attributes are dozens of ...genetic loci containing biosynthetic gene clusters (BGCs) producing bioactive small molecules (often referred to as secondary metabolites or natural products) that provide growth advantages to the fungus dependent on environment. Here we summarize the current knowledge of these BGCs—18 of which can be named to product—their expression profiles in vivo, and which BGCs may enhance virulence of this opportunistic human pathogen. Furthermore, we find extensive evidence for the presence of many of these BGCs, or similar BGCs, in distantly related genera including the emerging pathogen Pseudogymnoascus destructans, the causative agent of white-nose syndrome in bats, and suggest such BGCs may be predictive of pathogenic potential in other fungi.
This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.
We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri ...NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".
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