Aspergillus fumigatus is the most ubiquitous opportunistic filamentous fungal pathogen of human. As an initial step toward sequencing the entire genome of A. fumigatus, which is estimated to be approximately 30 Mb in size, we have sequenced a 922 kb region, contained within 16 overlapping bacterial artificial chromosome (BAC) clones. Fifty-four percent of the DNA is predicted to be coding with 341 putative protein coding genes. Functional classification of the proteins showed the presence of a higher proportion of enzymes and membrane transporters when compared to those of Saccharomyces cerevisiae. In addition to the nitrate assimilation gene cluster, the quinate utilisation gene cluster is also present on this 922 kb genomic sequence. We observed large scale synteny between A. fumigatus and Aspergillus nidulans by comparing this sequence to the A. nidulans genetic map of linkage group VIII.
"Aspergillus strains used in this study are displayed in Table S1 (references cited in the table , , , , , , , , , , , , , , , , , , , ). All fungal strains were maintained on solid glucose minimal media (GMM, 1 % glucose), potato dextrose agar (PDA), and Sabouraud dextrose media (SAB) including 1.5 % (w/v) agar as previously described at 30 °C in the dark. "
[Show abstract][Hide abstract] ABSTRACT: Conidiogenesis is the primary process for asexual reproduction in filamentous fungi. As the conidia resulting from the conidiogenesis process are primarily disseminated via air currents and/or water, an outstanding question has been how fungi recognize aerial environments suitable for conidial development. In this study, we documented the somewhat complex development of the conidia-bearing structures, termed conidiophores, from several Aspergillus species in a subsurface (gel-phase) layer of solid media. A subset of the isolates studied was able to develop conidiophores in a gel-phase environment, but exposure to the aeriform environment was required for the terminal developmental transition from phialide cells to conidia. The remaining Aspergilli could not initiate the conidiogenesis process until they were exposed to the aeriform environment. Our observations of conidiophore development in high or low oxygen conditions in both aeriform and gel-phase environments revealed that oxygen and the aeriform state are positive environmental factors for inducing conidiogenesis in most of the aspergilli tested in this study. Transcriptional analysis using A. fumigatus strain AF293 confined to either the aeriform or gel-phase environments revealed that expression of a key regulatory gene for conidiophore development (AfubrlA) is facilitated by oxygen while expression of another regulatory gene controlling conidia formation from phialides (AfuabaA) was repressed regardless of oxygen levels in the gel-embedded environment. Furthermore, by comparing the developmental behavior of conidiation-defective mutants lacking genes controlling various regulatory checkpoints throughout the conidiogenesis pathway, we propose that this aerial response by the fungus requires both oxygen and the phase transition (solid to aeriform), with these environmental signals integrating into the upstream regulatory pathway and central regulatory pathway of conidiogenesis, respectively. Our findings provide not only novel insight into how fungi respond to an aerial environment to trigger development for airborne conidia production but also the relationship between environmental factors and conidiogenesis regulation in aspergilli.
PLoS ONE 09/2013; 8(9):e74805. DOI:10.1371/journal.pone.0074805 · 3.23 Impact Factor
"Our first report in 2004 (8), presented data from a pilot study of clinical isolate Aspergillus fumigatus Af293 (9). A. fumigatus is the most common mould pathogen of humans, causing both life-threatening invasive diseases of immunocompromised patients and allergic disease in patients with atopic immune systems (3). "
[Show abstract][Hide abstract] ABSTRACT: The Central Aspergillus Data REpository (CADRE; http://www.cadre-genomes.org.uk) is a public resource for genomic data extracted from species of Aspergillus. It provides an array of online tools for searching and visualising features of this significant fungal genus. CADRE arose from a need within the medical community to understand the human pathogen Aspergillus fumigatus. Due to the paucity of Aspergillus genomic resources 10 years ago, the long-term goal of this project was to collate and maintain Aspergillus genomes as they became available. Since our first release in 2004, the resource has expanded to encompass annotated sequence for eight other Aspergilli and provides much needed support to the international Aspergillus research community. Recent developments, however, in sequencing technology are creating a vast amount of genomic data and, as a result, we shortly expect a tidal wave of Aspergillus data. In preparation for this, we have upgraded the database and software suite. This not only enables better management of more complex data sets, but also improves annotation by providing access to genome comparison data and the integration of high-throughput data.
"A. fumigatus H237, AF293, and CEA10 are clinical isolates , , . A. flavus (A1120, A1168, A249), A. nidulans (A4, A991, A92), and A. terreus A1156 strains were obtained from the Fungal Genetics Stock Center at the University of Missouri. Strains were maintained on and harvested from Aspergillus minimal medium (AMM) agar plates. "
[Show abstract][Hide abstract] ABSTRACT: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.
As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.
This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.
PLoS ONE 06/2011; 6(6):e21001. DOI:10.1371/journal.pone.0021001 · 3.23 Impact Factor
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