Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

Unlabelled: Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear.

Filamentous fungal-specific septin AspE is phosphorylated in vivo and interacts with actin, tubulin and other septins in the human pathogen Aspergillus fumigatus.

We previously analyzed the differential localization patterns of five septins (AspA-E), including a filamentous fungal-specific septin, AspE, in the human pathogen Aspergillus fumigatus. Here we utilized the A. fumigatus strain expressing an AspE-EGFP fusion protein and show that this novel septin with a tubular localization pattern in hyphae is phosphorylated in vivo and interacts with the other septins, AspA, AspB, AspC and AspD.

Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus.

Calcineurin, a heterodimer composed of the catalytic (CnaA) and regulatory (CnaB) subunits, plays key roles in growth, virulence and stress responses of fungi. To investigate the contribution of CnaA and CnaB to hyphal growth and septation, ΔcnaB and ΔcnaAΔcnaB strains of Aspergillus fumigatus were constructed. CnaA colocalizes to the contractile actin ring early during septation and remains at the centre of the mature septum.

The chitin synthase genes chsA and chsC are not required for cell wall stress responses in the human pathogen Aspergillus fumigatus.

Invasive aspergillosis is a leading cause of mortality in immunocompromised patients. The fungal cell wall is an attractive antifungal target, but it is dynamic and responsive to external stressors. The existence of multiple chitin synthases within Aspergilli is thought to reflect specialized functions in cell wall damage responses that facilitate continued growth and viability.

Differential localization patterns of septins during growth of the human fungal pathogen Aspergillus fumigatus reveal novel functions.

Septins, a conserved family of GTPases, are heteropolymeric filament-forming proteins that associate with the cell membrane and cytoskeleton and serve essential functions in cell division and morphogenesis. Their roles in fungal cell wall chitin deposition, septation, cytokinesis, and sporulation have been well established and they have recently been implicated in tissue invasion and virulence in Candida albicans. Septins have never been investigated in the human pathogenic fungus, Aspergillus fumigatus, which is a leading cause of death in immunocompromised patients.

Aspergillus oryzae type III polyketide synthase CsyA is involved in the biosynthesis of 3,5-dihydroxybenzoic acid.

As a novel superfamily of type III polyketide synthases in microbes, four genes csyA, csyB, csyC, and csyD, were found in the genome of Aspergillus oryzae, an industrially important filamentous fungus. In order to analyze their functions, we carried out the overexpression of csyA under the control of alpha-amylase promoter in A. oryzae and identified 3,5-dihydroxybenzoic acid (DHBA) as the major product.

Identification of csypyrone B1 as the novel product of Aspergillus oryzae type III polyketide synthase CsyB.

As a novel superfamily of type III polyketide synthases (PKSs) in microbes, four genes, csyA, csyB, csyC, and csyD, were found in the genome of Aspergillus oryzae, an industrially important filamentous fungus. Although orthologs of csyA, csyC, and csyD genes are present in a closely related species, Aspergillus flavus, csyB gene is unique to A. oryzae.

The Aspergillus fumigatus P-type Golgi apparatus Ca2+/Mn2+ ATPase PmrA is involved in cation homeostasis and cell wall integrity but is not essential for pathogenesis.

The Aspergillus fumigatus DeltapmrA (Golgi apparatus Ca(2+)/Mn(2+) P-type ATPase) strain has osmotically suppressible basal growth defects and cationic tolerance associated with increased expression of calcineurin pathway genes. Despite increased beta-glucan and chitin content, it is hypersensitive to cell wall inhibitors but remains virulent, suggesting a role for PmrA in cation homeostasis and cell wall integrity.

Functional expression of the Aspergillus flavus PKS-NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid.

alpha-Cyclopiazonic acid (CPA) is an indole tetramic acid mycotoxin. Based on our identification of the polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) hybrid gene cpaA involved in cyclopiazonic acid biosynthesis in Aspergillus fungi, we carried out heterologous expression of Aspergillus flavuscpaA under alpha-amylase promoter in Aspergillus oryzae and identified its sole product to be the CPA biosynthetic intermediate cyclo-acetoacetyl-l-tryptophan (cAATrp). This result rationalized that the PKS-NRPS hybrid enzyme CpaA catalyzes condensation of the diketide acetoacetyl-ACP formed by the PKS module and l-Trp activated by the NRPS module.

Aspergillus fumigatus calcipressin CbpA is involved in hyphal growth and calcium homeostasis.

Calcineurin is a conserved protein phosphatase that plays a critical role in Ca(2+) signaling and stress responses. Previously, a new class of conserved calcineurin-binding proteins, the calcipressins, was identified. However, the role of these proteins remains controversial, and both inhibitory and stimulatory effects on calcineurin were observed.

Disruption of the Aopex11-1 gene involved in peroxisome proliferation leads to impaired Woronin body formation in Aspergillus oryzae.

The Woronin body, a unique organelle found in the Pezizomycotina, plugs the septal pore upon hyphal damage to prevent excessive cytoplasmic bleeding. Although it was previously shown that the Woronin body buds out from the peroxisome, the relationship between peroxisomal proliferation/division and Woronin body differentiation has not been extensively investigated. In this report, we examined whether Pex11 required for peroxisomal proliferation participates in Woronin body formation in Aspergillus oryzae.

Differential effects of inhibiting chitin and 1,3-{beta}-D-glucan synthesis in ras and calcineurin mutants of Aspergillus fumigatus.

Aspergillus fumigatus must be able to properly form hyphae and maintain cell wall integrity in order to establish invasive disease. Ras proteins and calcineurin each have been implicated as having roles in these processes. Here, we further delineate the roles of calcineurin and Ras activity in cell wall biosynthesis and hyphal morphology using genetic and pharmacologic tools.

Calcineurin localizes to the hyphal septum in Aspergillus fumigatus: implications for septum formation and conidiophore development.

A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus DeltacnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.

Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae.

In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with alpha-amylase (AmyB) was expressed by A.

Analysis of expressed sequence tags from the fungus Aspergillus oryzae cultured under different conditions.

We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.

Phosphorylation of the Aspergillus oryzae Woronin body protein, AoHex1, by protein kinase C: evidence for its role in the multimerization and proper localization of the Woronin body protein.

Woronin body, a specialized peroxisome, is a unique organelle involved in septal pore sealing and protecting filamentous fungus from excessive cytoplasmic bleeding. We recently characterized the Aohex1 gene encoding the major protein of the Woronin body in the fungus Aspergillus oryzae. Although three-dimensional microscopy revealed plugging of the septal pore by Woronin body, the mechanism of its formation remains unknown.

Genomics of Aspergillus oryzae.

The genome sequence of Aspergillus oryzae, a fungus used in the production of the traditional Japanese fermentation foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste), has revealed prominent features in its gene composition as compared to those of Saccharomyces cerevisiae and Neurospora crassa. The A. oryzae genome is extremely enriched with genes involved in biomass degradation, primary and secondary metabolism, transcriptional regulation, and cell signaling.

Putative calmodulin-binding domains in aflatoxin biosynthesis-regulatory proteins.

The inhibition of aflatoxin production by trifluoperazine, an anticalmodulin (CaM) agent and the relevance of Ca(2+)/CaM-dependent phosphorylation and dephosphorylation during aflatoxin biosynthesis was previously reported. To identify proteins that may be regulated by CaM, an in silico analysis for putative CaM-binding domains (CaMBDs) in the aflatoxin-related proteins of Aspergillus parasiticus was performed using the CaM target database. Interestingly, the key regulators of aflatoxin biosynthesis such as AflR and AflJ contained predicted CaMBDs at their C-termini.

Genomics reveals traces of fungal phenylpropanoid-flavonoid metabolic pathway in the f ilamentous fungus Aspergillus oryzae.

Fungal secondary metabolites constitute a wide variety of compounds which either play a vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes.

Development of a modified positive selection medium that allows to isolate Aspergillus oryzae strains cured of the integrated niaD-based plasmid.

The nitrate reductase gene (niaD) is the most frequently utilized as a selectable marker for homologous integration at the niaD locus of Aspergillus oryzae. In this study we developed a method for curing of the niaD-based plasmid integrated on the A. oryzae genome.

Genome sequencing and analysis of Aspergillus oryzae.

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology.

Genomic evidences for the existence of a phenylpropanoid metabolic pathway in Aspergillus oryzae.

Plants interact with their environment by producing a diverse array of secondary metabolites. A majority of these compounds are phenylpropanoids and flavonoids which are valued for their medicinal and agricultural properties. The phenylpropanoid biosynthesis pathway proceeds with the basic C6-C3 carbon skeleton of phenylalanine, and involves a wide range of enzymes viz.

Identification and characterization of rns4/vps32 mutation in the RNase T1 expression-sensitive strain of Saccharomyces cerevisiae: Evidence for altered ambient response resulting in transportation of the secretory protein to vacuoles.

We previously reported a genetic analysis of the growth-inhibitory effect caused by the overexpression of the Aspergillus oryzae rntA gene, encoding RNase T1 (Ribonuclease T1), in Saccharomyces cerevisiae. Subsequently, rns (ribonuclease T1 sensitive) mutants with mutations in the rns1 (DSL1), rns2 (UMP1), and rns3 (SEC17) genes, were identified. In the present study, rns4 (VPS32/SNF7) gene mutation was identified by complementation of tunicamycin sensitivity.

Three-dimensional image analysis of plugging at the septal pore by Woronin body during hypotonic shock inducing hyphal tip bursting in the filamentous fungus Aspergillus oryzae.

We observed that the filamentous fungus, Aspergillus oryzae, grown on agar media burst out cytoplasmic constituents from the hyphal tip soon after flooding with water. Woronin body is a specialized organelle known to plug the septal pore adjacent to the lysed compartment to prevent extensive loss of cytoplasm. A.

Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae.

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein.