Evolution of asexual and sexual reproduction in the aspergilli

Aspergillus nidulans has long-been used as a model organism to gain insights into the genetic basis of asexual and sexual developmental processes both in other members of the genus Aspergillus, and filamentous fungi in general. Paradigms have been established concerning the regulatory mechanisms of conidial development. However, recent studies have shown considerable genome divergence in the fungal kingdom, questioning the general applicability of findings from Aspergillus, and certain longstanding evolutionary theories have been questioned. The phylogenetic distribution of key regulatory elements of asexual reproduction in A. nidulans was investigated in a broad taxonomic range of fungi. This revealed that some proteins were well conserved in the Pezizomycotina (e.g. AbaA, FlbA, FluG, NsdD, MedA, and some velvet proteins), suggesting similar developmental roles. However, other elements (e.g. BrlA) had a more restricted distribution solely in the Eurotiomycetes, and it appears that the genetic control of sporulation seems to be more complex in the aspergilli than in some other taxonomic groups of the Pezizomycotina. The evolution of the velvet protein family is discussed based on the history of expansion and contraction events in the early divergent fungi. Heterologous expression of the A. nidulans abaA gene in Monascus ruber failed to induce development of complete conidiophores as seen in the aspergilli, but did result in increased conidial production. The absence of many components of the asexual developmental pathway from members of the Saccharomycotina supports the hypothesis that differences in the complexity of their spore formation is due in part to the increased diversity of the sporulation machinery evident in the Pezizomycotina. Investigations were also made into the evolution of sex and sexuality in the aspergilli. MAT loci were identified from the heterothallic Aspergillus (Emericella) heterothallicus and Aspergillus (Neosartorya) fennelliae and the homothallic Aspergillus pseudoglaucus (=Eurotium repens). A consistent architecture of the MAT locus was seen in these and other heterothallic aspergilli whereas much variation was seen in the arrangement of MAT loci in homothallic aspergilli. This suggested that it is most likely that the common ancestor of the aspergilli exhibited a heterothallic breeding system. Finally, the supposed prevalence of asexuality in the aspergilli was examined. Investigations were made using A. clavatus as a representative ‘asexual’ species. It was possible to induce a sexual cycle in A. clavatus given the correct MAT1-1 and MAT1-2 partners and environmental conditions, with recombination confirmed utilising molecular markers. This indicated that sexual reproduction might be possible in many supposedly asexual aspergilli and beyond, providing general insights into the nature of asexuality in fungi.National Natural Science Foundation of China 31601446National Research Foundation of Korea 2016010945Intelligent Synthetic Biology Center of Global Frontier Projects 2015M3A6A8065838Biotechnology and Biological Sciences Research CouncilGovernment of IraqMinisterio de Economía y Competitividad BIO2015-67148-

Global sexual fertility in the opportunistic pathogen aspergillus fumigatus and identification of new supermater strains

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. A sexual cycle in Aspergillus fumigatus was first described in 2009 with isolates from Dublin, Ireland. However, the extent to which worldwide isolates can undergo sexual reproduction has remained unclear. In this study a global collection of 131 isolates was established with a near 1:1 ratio of mating types. All isolates were crossed to MAT1-1 or MAT1-2 Irish strains, and a subset of isolates from different continents were crossed together. Ninety seven percent of isolates were found to produce cleistothecia with at least one mating partner, showing that sexual fertility is not limited to the Irish population but is a characteristic of global A. fumigatus. However, large variation was seen in numbers of cleistothecia produced per cross, suggesting differences in the possibility for genetic exchange between strains in nature. The majority of crosses produced ascospores with >50% germination rates, but with wide variation evident. A high temperature heat shock was required to induce ascospore germination. Finally, a new set of highly fertile MAT1-1 and MAT1-2 supermater strains were identified and pyrimidine auxotrophs generated for community use. Results provide insights into the potential for the A. fumigatus sexual cycle to generate genetic variation and allow gene flow of medically important traits

First application of loop-mediated isothermal amplification (LAMP) assays for rapid identification of mating type in the heterothallic fungus Aspergillus fumigatus

Background Loop-mediated isothermal amplification (LAMP) assays, which operate at a single temperature and require no post-reaction processing, have been described for rapid species-specific detection of numerous fungi. The technology has much less commonly been applied to identification of other key genetic traits such as fungicide resistance, and has not yet been applied to mating-type determination in any fungus. Objectives To develop first LAMP assays for mating-type identification in a fungus, in this instance with the saprophytic mould and human opportunistic pathogen Aspergillus fumigatus, a heterothallic ascomycete requiring isolates of opposite mating type (MAT1-1, MAT1-2) for sexual reproduction. Methods New LAMP primer sets, targeted to MAT gene sequences, were screened against 34 A. fumigatus isolates (of known mating type) from diverse clinical, environmental and geographic sources to establish if they could distinguish MAT1-1 or MAT1-2 genotypes. Results and conclusions The new assays, operating at a single temperature of 65°C, correctly identified the mating-type of A. fumigatus isolates in <20 minutes, and thus have numerous research and practical applications. Similar MAT LAMP assays could now be developed for other fungi of agricultural, environmental, industrial and/or medical importance

Factors affecting germination of ascospores in Aspergillus fumigatus

Background and objective: Aspergillus fumigatus is the most common airborne fungal pathogen of humans. It is an opportunistic human pathogen in immunocompromised hosts. Following the recent discovery of the sexual reproductive cycle ascospores of this fungus were available and their germination was studied in the present study. This study aimed to study factors influencing germination of ascospores of A. fumigatus. Methods: A total of 12 environmental A. fumigatus were chosen in both mating type, examined for sexual reproduction by crossing complementary mating type, following removing of cleistothecia, preparation of ascospore suspensions in 0.05% Tween 20, then heat treated to kill any remaining conidia. Results: Ascospore germination of greater than 75% occurred at 28 °C on a wide variety of mycological media, 0.5% (ACM) was chosen for use in subsequent studies. Ascospores did not require a heat treatment prior to germination; however, heat treatment of ascospores at 75 °C for 1 hour previous to incubation enhanced germination to 89% and also served to inactivate contaminating conidia. Ascospores also exhibited a psychrotolerance following exposure at -80 °C for 96 hours. Inoculum concentration did not have a significant effect on germination rates between the range of 0.125×106 to 4×106 ascospores ml-1 (P >0.05). Conclusion: This study demonstrated that ascospore germination of greater than 75% occurred at 28 °C on a wide variety of mycological media. Ascospores of A. fumigatus are thermotolerant also exhibited a psychrotolerance whilst at the same time retaining viable sexual ascospores, to assess percentage germination a drop of Lactophenol Cotton Blue was added

Ecology of aspergillosis: insights into the pathogenic potency of <em>Aspergillus fumigatus</em> and some other <em>Aspergillus</em> species:insights into the pathogenic potency of <i>Aspergillus fumigatus </i>and some other <i>Aspergillus</i> species

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