Functional characterization of the Aspergillus fumigatusPHO80 homologue

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P_i) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P_i at normal or high external P_i concentrations) and a high-affinity (activated in response to P_i starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB^PHO80. We show that the ΔphoB^PHO80 mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB^PHO80, calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and ΔphoB^PHO80 mutant cells identify Afu4g03610 (phoD^PHO84), Afu7g06350 (phoE^PHO89), Afu4g06020 (phoC^PHO81), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the ΔphoB^PHO80 mutant background and under phosphate-limiting conditions of 0.1 mM P_i. Epifluorescence microscopy revealed accumulation of poly-phosphate in ΔphoB^PHO80 vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD^PHO84 deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, ΔphoB^PHO80 and ΔphoD^PHO84 mutant are fully virulent in a murine low dose model for invasive aspergillosis.     

The structures of polysaccharides and glycolipids of Aspergillus fumigatus grown in the presence of human serum

A study was made of polysaccharides and glycosphingolipids isolated from Aspergillus fumigatus grown in media supplemented with human serum from healthy donors. Fractionation of Cetavlon-precipitated polysaccharides on Sephacryl S-400 gave rise to an excluded fraction (Fraction I) with molecular weight of >400 kDa and an included peak (Fraction II) with an average molecular weight of 30–80 kDa. Fraction I comprises about 5% of total polysaccharide and was identified as a glycogen-like molecule. Its structure was deduced from methylation data, treatment with amyloglucosidase, a red-brown coloration produced with an iodine solution and by ¹H and ¹³C-NMR spectroscopy. It was previously suggested that higher amounts of glycogen-like polysaccharide (20%) were present in A. fumigatus grown in serum-free medium. Fraction II was identified as a galactomannan and was the main polysaccharide of A. fumigatus grown in serum-supplemented medium. Its structure was elucidated mainly by ¹³C-NMR spectroscopy combined with partial acetolysis and methylation analysis. The ¹³C-NMR spectrum of the galactomannan showed a much greater complexity in the -d-galf and -d-manp C-1 regions, than was evident for galactomannan from serum-free cultures previously described, reflecting differences in the glycosylation pattern, stimulated in serum-supplemented medium.No differences in A. fumigatus glycosphingolipid could be detected between serum-containing and serum-free growth conditions.Our results demonstrate that the change in polysaccharide structure is a more specific response to the altered growth conditions and not merely a symptom of more general changes.This revised version was published online in October 2005 with corrections to the Cover Date.     

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Two common fungi associated with farmer's lung: Fine structure of Aspergillus fumigatus and Aspergillus umbrosus

The fine structure of Aspergillus fumigatus and Aspergillus umbrosus by transmission electron microscopy (TEM) is described. The fine structure of the ascosporic and asexual stages of A. umbrosus is described for the first time. Dense, homogenous material and fibers were detected on the outer hyphal cell wall of the Aspergilli. Septal pores were found in the hypha of A. umbrosus. Two wall layers were detected in the cell wall of the conidia of the both Aspergilli. The ascospores of A. umbrosus contained thick cell wall and the surface of which was smoother than that of the conidia.     

Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus

Aspergillus fumigatus (ATCC 28282), a thermotolerant fungus, has been shown to be capable of growth on phenol as the sole carbon and energy source. During growth of the organism on phenol, catechol and hydroquinone accumulated transiently in the medium; cells grown on phenol oxidised these compounds without a lag period. Two different routes operating simultaneously, leading to different ring-fission substrates, are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol, which is then cleaved by an intradiol mechanism leading to 3-oxoadipate. In the other route, phenol is hydroxylated in the para-position to produce hydroquinone, which is then converted into 1,2,4-trihydroxybenzene for ring fission by ortho-cleavage to give maleylacetate. Cell-free extracts of phenol-grown mycelia were found to contain enzymic activities for the proposed steps. Two ring-fission dioxygenases, one active towards 1,2,4-trihydroxybenzene, but not catechol, and one active towards both ring-fission substrates, were separated by FPLC. Succinate-grown mycelia did not oxidise any of the intermediates until a clear lag period had elapsed and did not contain any of the enzymic activities for phenol metabolism.     

Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus

When the opportunistic pathogen Aspergillus fumigatus entered the stationary phase, there was a rapid loss in cell viability which was associated with the appearance of markers characteristic of apoptosis, namely annexin V-FITC binding to the cytoplasmic membrane, demonstrating exposure of phosphatidylserine to the outer leaflet of the membrane; and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining of the nuclei, indicating DNA fragmentation. This was followed later by a loss of membrane integrity as revealed by propidium iodide staining. The development of the apoptotic phenotype was blocked when the protein synthesis inhibitor cycloheximide was added to the culture 1h prior to the onset of the stationary phase, demonstrating active participation of the cell. In addition, intracellular activity against substrates specific for caspase-1 and -8 also increased on stationary phase entry and the development of the apoptotic phenotype was blocked when the cell permeant caspase inhibitor Z-FAD-fmk was present in the medium. Cell death in A. fumigatus during the stationary phase therefore appears to share similarities to apoptotic cell death in higher eukaryotes and to be dependent on a caspase-like activity.     

Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis

Summary Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the -galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.     

Silencing of mitochondrial alternative oxidase gene of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.     

The appearance of an enzyme activity catalysing the conversion of norsolorinic acid to averantin in Aspergillus parasiticus cultures

The activity of the enzyme responsible for the conversion of norsolorinic acid to averantin was studied in two strains of Aspergillus parasiticus. Cell-free extracts of the enzyme were purified from different aged mycelia and little activity was found prior to 24 hours after inoculation but this quickly reached a maximum at 48 hours and declined thereafter. Both strains of A. parasiticus, one in aflatoxin producing strain, the other a versicolorin A accumulating mutant, showed this trend. It was concluded that the enzyme responsible for this conversion was a secondary metabolic enzyme and was distinct from alcohol and mannitol dehydrogenases.     

Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans

Summary Acetate inducible genes of Aspergillus nidulans were cloned via differential hybridization to cDNA probes. Using transformation of mutant strains the genes were identified as facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase). The levels of RNA encoded by these genes were shown to be acetate inducible and subject to carbon catabolite repression. Induction is abolished in a facB mutant and carbon catabolite repression is relieved in a creA mutant.     

Induction of systemic acquired resistance in Zea mays L. by Aspergillus flavus and A. parasiticus derived elicitors

Host defence mechanisms can be elicited by using different elicitors produced from the pathogen/host. In this study, an effort has been made to study the effect of two fungal elicitors derived from Aspergillus flavus and A. parasiticus on induction of various defence-related enzymes in maize (Zea mays L.). Foliar application was done on 20-days-old maize plant with 10% A. flavus fungal culture filtrate (AFFCF) and A. parasiticus fungal culture filtrate (APFCF) as elicitors to trigger systemic acquired resistance (SAR). As a response of SAR, an increase in activities of phenylalanine ammonia lyase (PAL), peroxidase (POX), β-1,3-glucanase, nitrate reductase (NR) and nitrite reductase (NiR), total proteins were found highest on 4th day after treatment (DAT), whereas total carbohydrate and total chlorophyll on 2nd and 6th DAT, respectively, in comparison with the control plants. The SDS PAGE analysis revealed the induction of PR proteins, namely Chitinase (25, 29?kDa) and β-1,3-glucanase (33?kDa), in treated plants in comparison with untreated control plants. The treated plants showed enhanced growth and development as well as increase in yield. About 100% survival rate was found in maize seeds treated with AFFCF and APFCF and grown on respective fungal infested soil than control. The enhanced activities of defence enzymes and elevated protein, carbohydrate, chlorophyll content in treated maize plants suggest the induction of SAR against A. flavus and A. parasiticus by using the same fungal elicitors.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3^– null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Functional conversion of fatty acyl-CoA synthetase to firefly luciferase by site-directed mutagenesis: A key substitution responsible for luminescence activity

We demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis [Oba, Y., Ojika, M. and Inouye, S. (2003) Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett. 540, 251-254] and proposed that the evolutionary origin of beetle luciferase is a fatty acyl-CoA synthetase (FACS) in insect. In this study, we performed the functional conversion of FACS to luciferase by replacing a single amino acid to serine. This serine residue is conserved in luciferases and possibly interacts with luciferin. The mutants of FACSs in non-luminous click beetle Agrypnus binodulus (AbLL) and Drosophilamelanogaster (CG6178) gave luminescence enhancement, suggesting that the serine residue is a key substitution responsible for luminescence activity.     

Coffee husk: an inexpensive substrate for production of citric acid by Aspergillus niger in a solid-state fermentation system

Aspergillus niger CFTRI 30 produced 1.3 g citric acid/10 g dry coffee husk in 72 h solid-state fermentation when the substrate was moistened with 0.075 M NaOH solution. Production was increased by 17% by adding a mixture of iron, copper and zinc to the medium but enrichment of the moist solid medium with (NH₄)₂SO₄, sucrose or any of four enzymes did not improve production. The production of about 1.5 g citric acid/10 g dry coffee husk at a conversion of 82% (based on sugar consumed) under standardized conditions demonstrates the commercial potential of using the husk in this way.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India;     

A common class of nematode glutathione S-transferase (GST) revealed by the theoretical proteome of the model organism Caenorhabditis elegans

The genome verified C. elegans free-living nematode model is a new tool for investigating gene expression in human and animal nematode parasites. There is limited information on designating glutathione S-transferase (GST) to specific classes in lower invertebrates such as nematodes. Following cloning, amino acid sequence alignment, recombinant expression and Western blotting we provide evidence of a new GST class in nematodes or lower invertebrates.     

The light-dependent regulator velvet A of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> acts as a repressor of the penicillin biosynthesis

The biosynthesis of the β-lactam antibiotic penicillin in Aspergillus nidulans is catalysed by three enzymes that are encoded by the genes acvA, ipnA and aatA. Several studies have indicated that these genes are controlled by a complex regulatory network, including a variety of cis-acting DNA elements and regulatory factors. Until now, however, relatively little information is available on external signals and their transmission influencing the expression of the structural genes. Here, we show that the light-dependent regulator velvet A (VeA) acts as a repressor on the penicillin biosynthesis, mainly via repression of the acvA gene. Expression of a regulatable alcAp-veA gene fusion in an A. nidulans strain carrying, in addition, acvAp-uidA and ipnAp-lacZ gene fusions indicated that under alcAp-inducing conditions, penicillin titres and expression of acvAp-uidA were drastically reduced compared with untransformed wild-type strains. The same level of repression was found irrespective of whether the alcAp-veA gene fusion was expressed in a veA1 or ΔveA background, with or without light. The expression of the ipnAp-lacZ gene fusion was only moderately affected indicating a less prominent effect. These findings were confirmed by the analysis of a regulatable niiAp-veA gene fusion. Under niiAp-inducing conditions, penicillin titres and acvAp-uidA expression were much lower than in untransformed wild-type strains.     

Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production

Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL^–1), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy d-glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.