Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses.

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K-means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high-adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin-like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.

Fucose-specific lectin of Aspergillus fumigatus: binding properties and effects on immune response stimulation.

Aspergillus fumigatus is the major causative fungus of aspergillosis, and many studies have explored the relationship between A. fumigatus and pathogenicity. In the current study, we focused on a fucose-specific lectin, FleA, as a novel molecule which related to the pathogenicity of A. fumigatus. The disruption of the fleA gene did not lead to clear morphological changes compared to parental strain under several stress conditions in culture, but germination become earlier. In comparison with parental strain, the pathogenicity of disruptant was enhanced in a mouse infection model. The pattern of conidial phagocytosis and adhesion to cultured cells did not explain this enhanced pathogenicity. FleA was reported to contain six conserved fucose-binding sites; the analysis of constructed FleA point mutants revealed nonequivalent contribution of the fucose-binding sites to fucose binding. Based on the immune response induced in the cultured cells upon exposure to wild-type and mutant FleA, we propose a model of the FleA molecule in A. fumigatus infection.

Global gene expression reveals stress-responsive genes in Aspergillus fumigatus mycelia.

BACKGROUND: Aspergillus fumigatus is a human fungal pathogen that causes aspergillosis in immunocompromised hosts. A. fumigatus is believed to be exposed to diverse environmental stresses in the host cells. The adaptation mechanisms are critical for infections in human bodies. Transcriptional networks in response to diverse environmental challenges remain to be elucidated. To gain insights into the adaptation to environmental stresses in A. fumigatus mycelia, we conducted time series transcriptome analyses. RESULTS: With the aid of RNA-seq, we explored the global gene expression profiles of mycelia in A. fumigatus upon exposure to diverse environmental changes, including heat, superoxide, and osmotic stresses. From the perspective of global transcriptomes, transient responses to superoxide and osmotic stresses were observed while responses to heat stresses were gradual. We identified the stress-responsive genes for particular stresses, and the 266 genes whose expression levels drastically fluctuated upon exposure to all tested stresses. Among these, the 77 environmental stress response genes are conserved in S. cerevisiae, suggesting that these genes might be more general prerequisites for adaptation to environmental stresses. Finally, we revealed the strong correlations among expression profiles of genes related to 'rRNA processing'. CONCLUSIONS: The time series transcriptome analysis revealed the stress-responsive genes underlying the adaptation mechanisms in A. fumigatus mycelia. These results will shed light on the regulatory networks underpinning the adaptation of the filamentous fungi.

Transcription factor Afmac1 controls copper import machinery in Aspergillus fumigatus.

Copper (Cu) is an essential metal for all living organisms, although it is toxic in excess. Filamentous fungus must acquire copper from its environment for growth. Despite its essentiality for growth, the mechanisms that maintain copper homeostasis are not fully understood in filamentous fungus. To gain insights into copper homeostasis, we investigated the roles of a copper transcription factor Afmac1 in the life-threatening fungus Aspergillus fumigatus, a homolog of the yeast MAC1. We observed that the Afmac1 deletion mutant exhibited not only significantly slower growth, but also incomplete conidiation including a short chain of conidia and defective melanin. Moreover, the expressions of the copper transporters, ctrA1, ctrA2, and ctrC, and metalloreductases, Afu8g01310 and fre7, were repressed in ∆Afmac1 cells, while those expressions were induced under copper depletion conditions in wild-type. The expressions of pksP and wetA, which are, respectively, involved in biosynthesis of conidia-specific melanin and the late stage of conidiogenesis, were decreased in the ∆Afmac1 strain under minimal media condition. Taken together, these results indicate that copper acquisition through AfMac1 functions in growth as well as conidiation.

Nabscessins A and B, Aminocyclitol Derivatives from Nocardia abscessus IFM 10029T.

Two new aminocyclitol amide derivatives, nabscessins A (1) and B (2), were isolated from the culture broth of a pathogenic actinomycete species, Nocardia abscessus IFM 10029T. The structures of nabscessins A and B were elucidated by spectral studies, and the compounds showed antifungal activity against Cryptococcus neoformans, with IC50 values of 32 and 16 µg/mL, respectively.

Zamamidine D, a Manzamine Alkaloid from an Okinawan Amphimedon sp. Marine Sponge.

A new manzamine alkaloid, zamamidine D (1), was isolated from an Okinawan Amphimedon sp. marine sponge. The structure of zamamidine D (1) including the relative configuration was elucidated on the basis of spectroscopic data. Zamamidine D (1) is the first manzamine alkaloid possessing a 2,2'-methylenebistryptamine unit as the aromatic moiety instead of a ß-carboline unit. Zamamidine D (1) showed antimicrobial activity against several bacteria and fungi.

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

Hyrtinadines C and D, New Azepinoindole-Type Alkaloids from a Marine Sponge Hyrtios sp.

New bisindole alkaloids, hyrtinadines C (1) and D (2), have been isolated from an Okinawan marine sponge Hyrtios sp. The structures of hyrtinadines C (1) and D (2) were elucidated based on analyses of the spectral data. Hyrtinadines C (1) and D (2) were the relatively rare alkaloids possessing a 3,4-fused azepinoindole skeleton. Hyrtinadines C (1) and D (2) showed antimicrobial activity.

Genome sequence comparison of Aspergillus fumigatus strains isolated from patients with pulmonary aspergilloma and chronic necrotizing pulmonary aspergillosis.

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus.

Tyrokeradines G and H, new bromotyrosine alkaloids from an Okinawan Verongid sponge.

Two new bromotyrosine alkaloids, tyrokeradines G (1) and H (2), have been isolated from an Okinawan marine sponge of the order Verongida. The structures of 1 and 2 were elucidated on the basis of spectroscopic data. Tyrokeradine G (1) is the first bromotyrosine alkaloid possessing a ß-alanine unit, while tyrokeradine H (2) is a rare bromotyrosine alkaloid possessing a N-substituted pyridinium ring. Tyrokeradines G (1) and H (2) showed antifungal activity.

Prenylated benzophenones from Triadenum japonicum.

Six new prenylated benzophenones, (-)-nemorosonol (1) and trijapins A-E (2-6), were isolated from the aerial parts of Triadenum japonicum. (-)-Nemorosonol (1) and trijapins A-C (2-4) have a common tricyclo[4.3.1.0(3,7)]decane skeleton, while 1 is an enantiomer of (+)-nemorosonol previously isolated from Clusia nemorosa. The absolute configuration of (-)-nemorosonol (1) was assigned by ECD spectroscopy. Trijapins A-C (2-4) are analogues of 1 possessing an additional tetrahydrofuran ring. Trijapins D (5) and E (6) are prenylated benzophenones with a 1,2-dioxane moiety and a hydroperoxy group, respectively. (-)-Nemorosonol (1) exhibited antimicrobial activity against Escherichia coli (MIC, 8 µg/mL), Staphylococcus aureus (MIC, 16 µg/mL), Bacillus subtilis (MIC, 16 µg/mL), Micrococcus luteus (MIC, 32 µg/mL), Aspergillus niger (IC50, 16 µg/mL), Trichophyton mentagrophytes (IC50, 8 µg/mL), and Candida albicans (IC50, 32 µg/mL), while trijapin D (5) showed antimicrobial activity against C. albicans (IC50, 8 µg/mL).

Cyanide-bridged decanuclear cobalt-iron cage.

A cyanide-bridged decanuclear [Co6Fe4] cluster was synthesized by a one-pot reaction, and the magnetic properties and electronic configuration were investigated. The complex displayed thermally controlled electron-transfer-coupled spin transition (ETCST) behavior between Co(III) low-spin-NC-Fe(II) low-spin and Co(II) high-spin-NC-Fe(III) low-spin states, as confirmed by single-crystal X-ray, magnetic, and Mössbauer analyses.

Identification of fungal pathogens by visible microarray system in combination with isothermal gene amplification.

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 10(3) CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.

Subcomponents in humic acid structure contribute to the differential responses of Aspergillus oryzae strains to humic acid.

Humic acid (HA) is a complex natural organic macromolecule, can be decomposed to low-molecular compounds by some soil fungi and then influences the growth of fungi. Aspergillus oryzae is a fungus domesticated from its ancestor, which was supposed to live in soil. Group 3 strains of A. oryzae hold fewer aflatoxin-biosynthetic genes than group 1 strains and may differently response to HA because of the deletion of some genes along with the domestication. However, effect of HA on growth of A. oryzae group 1 and group 3 strains remains unclear. In this study, four strains of A. oryzae in group 1 and four in group 3 were point inoculated on equivalent medium (pH 7.3) with two commercially available HAs. The growth of RIB40 was the most stimulated among group 1 strains and that of RIB143 was the most inhibited among group 3 strains. To identify the basis of these differences, we examined the possible effects of HA subcomponents including polyphenol and minerals on the growth of RIB40 and RIB143. Polyphenol represented by gallic acid (GA), a partial structure common with model HA, and mineral ions including Al 3+ , Ca 2+ , Ti 4+ , Mn 2+ , Sr 2+ , and Ba2+ contributed to stimulating the growth of RIB40, whereas these components generally did not affect the growth of RIB143. Thus, our findings indicate that the sub-compositions of HAs, including GA and several minerals, were the main factors driving the different responses of RIB40 and RIB143 to HAs.

Comparative Genome Analysis of Japanese Field-Isolated Aspergillus for Aflatoxin Productivity and Non-Productivity.

Aspergillus flavus produces aflatoxin, a carcinogenic fungal toxin that poses a threat to the agricultural and food industries. There is a concern that the distribution of aflatoxin-producing A. flavus is expanding in Japan due to climate change, and it is necessary to understand what types of strains inhabit. In this study, we sequenced the genomes of four Aspergillus strains isolated from agricultural fields in the Ibaraki prefecture of Japan and identified their genetic variants. Phylogenetic analysis based on single-nucleotide variants revealed that the two aflatoxin-producing strains were closely related to A. flavus NRRL3357, whereas the two non-producing strains were closely related to the RIB40 strain of Aspergillus oryzae, a fungus widely used in the Japanese fermentation industry. A detailed analysis of the variants in the aflatoxin biosynthetic gene cluster showed that the two aflatoxin-producing strains belonged to different morphotype lineages. RT-qPCR results indicated that the expression of aflatoxin biosynthetic genes was consistent with aflatoxin production in the two aflatoxin-producing strains, whereas the two non-producing strains expressed most of the aflatoxin biosynthetic genes, unlike common knowledge in A. oryzae, suggesting that the lack of aflatoxin production was attributed to genes outside of the aflatoxin biosynthetic gene cluster in these strains.

Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae.

Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

Visual analysis of DNA microarray data for accurate molecular identification of non-albicans Candida isolates from patients with candidemia episodes.

The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance.

Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites.

Fungal secondary metabolites have been considered promising resources in the search for novel bioactive compounds. Given the high potential of fungi as genetic resources, it is essential to find an efficient way to link biosynthetic genes to the product in a heterologous system, because many genes for the secondary metabolite in the original strain are silent under standard laboratory conditions. In a previous study, we constructed a heterologous expression system for a biosynthetic gene cluster using Aspergillus oryzae as the host. To make the host more versatile for the expression of secondary metabolism genes, the expression levels of a global regulator, laeA, were increased by placing the A. oryzae laeA gene under the control of the constitutive active pgk promoter. In the A. oryzae overexpressing laeA, two clusters of heterologous biosynthetic genes [the monacolin K (MK) gene cluster from Monascus pilosus and the terrequinone A (TQ) gene cluster from Aspergillus nidulans] were successfully overexpressed, resulting in the production of the corresponding metabolite, MK or TQ. The successful production of secondary metabolites belonging to different structural groups, namely MK as a polyketide and TQ as a hybrid of amino acid and isoprenoid, indicated that the laeA-enriched A. oryzae was a versatile host for the heterologous expression of the biosynthetic gene cluster.

Phenotypic and Molecular Biological Analysis of Polymycovirus AfuPmV-1M From Aspergillus fumigatus: Reduced Fungal Virulence in a Mouse Infection Model.

The filamentous fungal pathogen Aspergillus fumigatus is one of the most common causal agents of invasive fungal infection in humans; the infection is associated with an alarmingly high mortality rate. In this study, we investigated whether a mycovirus, named AfuPmV-1M, can reduce the virulence of A. fumigatus in a mouse infection model. AfuPmV-1M has high sequence similarity to AfuPmV-1, one of the polymycovirus that is a capsidless four-segment double-stranded RNA (dsRNA) virus, previously isolated from the genome reference strain of A. fumigatus, Af293. However, we found the isolate had an additional fifth dsRNA segment, referred to as open reading frame 5 (ORF5), which has not been reported in AfuPmV-1. We then established isogenic lines of virus-infected and virus-free A. fumigatus strains. Mycovirus infection had apparent influences on fungal phenotypes, with the virus-infected strain producing a reduced mycelial mass and reduced conidial number in comparison with these features of the virus-free strain. Also, resting conidia of the infected strain showed reduced adherence to pulmonary epithelial cells and reduced tolerance to macrophage phagocytosis. In an immunosuppressed mouse infection model, the virus-infected strain showed reduced mortality in comparison with mortality due to the virus-free strain. RNA sequencing and high-performance liquid chromatography (HPLC) analysis showed that the virus suppressed the expression of genes for gliotoxin synthesis and its production at the mycelial stage. Conversely, the virus enhanced gene expression and biosynthesis of fumagillin. Viral RNA expression was enhanced during conidial maturation, conidial germination, and the mycelial stage. We presume that the RNA or translation products of the virus affected fungal phenotypes, including spore formation and toxin synthesis. To identify the mycovirus genes responsible for attenuation of fungal virulence, each viral ORF was ectopically expressed in the virus-free KU strain. We found that the expression of ORF2 and ORF5 reduced fungal virulence in the mouse model. In addition, ORF3 affected the stress tolerance of host A. fumigatus in culture. We hypothesize that the respective viral genes work cooperatively to suppress the pathogenicity of the fungal host.

Identification of mokB involved in monacolin K biosynthesis in Monascus pilosus.

Monacolin K (MK), which is widely used as an antihypercholesterolemia medicine, is produced as a fungal secondary metabolite through the polyketide pathway. The MK biosynthetic gene cluster proposed for Monascus pilosus BCRC38072 was also identified in M. pilosus NBRC4480. The mokB gene, located at the end of the putative gene cluster and possibly encoding polyketide synthase, was disrupted. The mokB disruptant did not produce MK, but accumulated an intermediate that was confirmed to be monacolin J, indicating that mokB encodes the polyketide synthase responsible for the biosynthesis of side-chain diketide moiety.