The role of thiol species in the hypertolerance of Aspergillus sp. P37 to arsenic

Aspergillus sp. P37 is an arsenate-hypertolerant fungus isolated from a river in Spain with a long history of contamination with metals. This strain is able to grow in the presence of 0.2 M arsenate, i.e. 20-fold higher than the reference strain, Aspergillus nidulans TS1. Although Aspergillus sp. P37 reduces As(V) to As(III), which is slowly pumped out of the cell, the measured efflux of oxyanions is insufficient to explain the high tolerance levels of this strain. To gain an insight into this paradox, the accumulation of acid-soluble thiol species in Aspergillus sp. P37 when exposed to arsenic was compared with that of the arsenic-sensitive A. nidulans TS1 strain. Increasing levels of arsenic in the medium did not diminish the intracellular pool of reduced glutathione in Aspergillus sp. P37, in sharp contrast with the decline of glutathione in A. nidulans under the same conditions. Furthermore, concentrations of arsenic that were inhibitory for the sensitive A. nidulans strain (e.g. 50 mM and above) provoked a massive formation of vacuoles filled with thiol species. Because the major fraction of the cellular arsenic was present as the glutathione conjugate As(GS)3, it is plausible that the arsenic-hypertolerant phenotype of Aspergillus sp. P37 is in part due to an enhanced capacity to maintain a large intracellular glutathione pool under conditions of arsenic exposure and to sequester As(GS)3 in vacuoles. High pressure liquid chromatography analysis of cell extracts revealed that the contact of Aspergillus sp. P37 (but not A. nidulans) with high arsenic concentrations (> or =150 mM) induced the production of small quantities of a distinct thiol species indistinguishable from plant phytochelatin-2. Yet, we argue that phytochelatins do not explain arsenic resistance in Aspergillus, and we advocate the role of As(GS)3 complexes in arsenic detoxification.     

Arsenate transport and reduction in the hyper-tolerant fungus Aspergillus sp. P37

Aspergillus sp. P37 is able to grow at arsenate concentrations of 0.2 M--more than 20-fold higher than that withstood by reference microorganisms such Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. This paper examines the transport of arsenate and phosphate and the reduction of arsenate in Aspergillus sp. P37. These properties were compared with the corresponding properties of the archetype strain Aspergillus nidulans TS1. Both uptake and efflux of arsenate were inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, suggesting that the transport system(s) is(are) membrane-potential dependent. As uptake of arsenate and phosphate are higher in Aspergillus sp. P37 than in A. nidulans, the increase in arsenate resistance cannot be accounted for by a change in uptake. Cells of both strains loaded with arsenic slowly released the oxyanion. Speciation of the arsenic in the medium showed an enhanced level of arsenate reduction in Aspergillus sp. P37. These data suggest that increased arsenate reduction is at least in part responsible for the hyper-tolerant phenotype of this fungus.     

Osmotic stress limits arsenic hypertolerance in Aspergillus sp. P37

Aspergillus sp. P37 is an arsenate-resistant fungus isolated from a river with a long history of heavy metal contamination. Its hypertolerant mechanism relies on an efficient arsenate reduction system in conjunction with increased coupling capabilities to glutathione and translocation of these complexes into the vacuolar system. Here, the comparison of arsenic resistance in two Aspergillus species and Saccharomyces cerevisiae in the presence or absence of different osmolytes revealed a link between tolerance to osmotic stress and tolerance to arsenic in Aspergillus strains. Under osmotic stress generated by NaCl or KCl, both Aspergillus strains were less resistant to arsenite and arsenate. In the presence of sorbitol, Aspergillus strains were more resistant to arsenite. Intracellular accumulation of arsenic was affected by the presence of the osmolytes (NaCl or sorbitol), suggesting a major role for vacuoles in the detoxification of arsenic in Aspergillus.     

Role of Aspergillus niger acrA in arsenic resistance and its use as the basis for an arsenic biosensor

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 μg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 μg/liter).     

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.     

Influence of Arsenic from Anthropogenic Loaded Soils on the Mine Water Quality in the Tin District Ehrenfriedersdorf,Erzgebirge (Germany)

The central part of the tin ore deposit Ehrenfriedersdorf/Erzgebirge, which was exploited from the 13^th century to 1990, was flooded from 1994 to 1996. Since that time mine waters have flown through the gallery “Tiefer Sauberger Stolln” to the creek Wilisch in the Elbe river catchment area. The water at the mine portal shows high concentrations of arsenic and heavy metals. The average arsenic concentration is about 0.5 mg/L. Approximately two thirds of arsenic are transported dissolved. Where the mine water ascends from deeper levels, arsenic concentrations of about 0.4 mg/L were found. Here arsenic occurs predominantly particular. The mining gallery “Tiefer Sauberger Stolln” provides the unique opportunity of subsurface sampling for the identification of the arsenic sources under different hydrological conditions (normal and high water level). The sources of dissolved arsenic in the gallery part between the raise and the portal were determined and analyzed. Between these two monitoring points, many inflows of infiltration water were detected. The concentration of As in the infiltration water reaches up to 1.8 mg/L, which varies depending on the location in the gallery and the hydrological situation. The first part of the gallery was straightened, heightened and partly concreted with modern mining technique. The arsenic concentrations can decrease owing to high precipitation rates and snow melt events. The last part of the gallery was preserved due to low coverage. Here the arsenic concentrations in the infiltration waters increase with the surface water inflow. At a normal water level, 1 kg arsenic per day leaves the raise and 2.1 kg the gallery portal, which means that 50 % of the arsenic load comes from the infiltration water. At a high water level, 2.5 kg arsenic per day are transported through the raise and 8.2 kg per day through the gallery portal, which means that about 70 % of the arsenic load comes from infiltration water. The area of Ehrenfriedersdorf is characterized by a superposition of anthropogenic soil pollution over the geogenic inventory. There is a close connection between ancient soil contaminations by high amounts of water‐soluble arsenic compounds, e.g. arsenic trioxide formed by roasting the ores during ancient tin smelting, and high concentrations of dissolved arsenic in the infiltration water. The contamination of surface water and river sediments by arsenic is originating from an anthropogenic pollution of soils by ancient tailings via infiltration of water rich in arsenic into the mine gallery.     

Let your enemy do the work: within-host interactions between two fungal parasites of leaf-cutting ants

Within-host competition is an important factor in host-parasite relationships, yet most studies consider interactions involving only single parasite species. We investigated the interaction between a virulent obligate entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, and a normally avirulent, opportunistic fungal pathogen, Aspergillus flavus, in their leaf-cutting ant host, Acromyrmex echinatior. Surprisingly, the latter normally out-competed the former in mixed infections and had enhanced fitness relative to when infecting in isolation. The result is most probably due to Metarhizium inhibiting the host's immune defences, which would otherwise normally prevent infections by Aspergillus. With the host defences negated by the virulent parasite, the avirulent parasite was then able to out-compete its competitor. This result is strikingly similar to that seen in immunocompromised vertebrate hosts and indicates that avirulent parasites may play a more important role in host life histories than is generally realized.     

Antifungal activity of the local complement system in cerebral aspergillosis

Dissemination of aspergillosis into the central nervous system is associated with nearly 100% mortality. To study the reasons for the antifungal immune failure we analyzed the efficacy of cerebral complement to combat the fungus Aspergillus. Incubation of Aspergillus in non-inflammatory cerebrospinal fluid (CSF) revealed that complement levels were sufficient to obtain a deposition on the surface, but opsonization was much weaker than in serum. Consequently complement deposition from normal CSF on fungal surface stimulated a very low phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by conidia opsonized in serum. Similarly, opsonization of Aspergillus by CSF was not sufficient to induce an oxidative burst in infiltrating granulocytes, whereas conidia opsonized in serum induced a clear respiratory signal. Thus, granulocytes were capable of considerably reducing the viability of serum-opsonized Aspergillus conidia, but not of conidia opsonized in CSF. The limited efficacy of antifungal attack by cerebral complement can be partly compensated by enhanced synthesis, leading to elevated complement concentrations in CSF derived from a patient with cerebral aspergillosis. This inflammatory CSF was able to induce (i) a higher complement deposition on the Aspergillus surface than non-inflammatory CSF, (ii) an accumulation of complement activation products and (iii) an increase in phagocytic and killing activity of infiltrating granulocytes. However, levels and efficacy of the serum-derived complement were not reached. These data indicate that low local complement synthesis and activation may represent a central reason for the insufficient antifungal defense in the brain and the high mortality rate of cerebral aspergillosis.     

Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii

The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii. This is the first report of a hemicellulase gene from this novel source. At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids. The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa. The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases. Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside. D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations. The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.     

Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe.

In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p/Nuf1p facilitates mitotic spindle formation from the fungal centrosome or spindle pole body (SPB). The human Spc110p orthologue kendrin is a centrosomal, calmodulin-binding pericentrin isoform that is specifically overexpressed in carcinoma cells. Here we establish an evolutionary and functional link between Spc110p and kendrin through identification and analysis of similar calmodulin-binding proteins in the fission yeast Schizosaccharomyces pombe (Pcp1p, pole target of calmodulin in S. pombe) and the filamentous fungus Aspergillus nidulans. Like Spc110p and kendrin, Pcp1p and the A. nidulans protein contain predicted coiled-coil secondary structure and a COOH-terminal calmodulin-binding region. Green fluorescent protein fusions of Pcp1p localize to the SPB as analyzed by fluorescence and immunoelectron microscopy. Pcp1p overexpression causes chromosome missegregation, multiple mitotic spindle fragments, and multiple abnormal SPB-like structures, a phenotype remarkably similar to that of many human carcinoma lines, which exhibit chromosome and spindle defects, and supernumerary centrosomes.     

印楝内生真菌棘孢曲霉YM 311498的代谢产物研究

从印楝植物内生真菌棘孢曲霉（Aspergillus aculeatus）YM311498的发酵液提取物中分离得到4个化合物,通过波谱技术分别鉴定为大黄酚（1）、大黄素（2）、琥珀酸（3）、柠檬酸二甲酯（4）,以上化合物均为首次从该种真菌中分离得到。     

Aspergillus in the Papanicolaou stain: morphology, fluorescence and diagnostic feasibility

hettlich c., küpper th., wehle k. and pfitzer p. (1998) Cytopathology 9, 381–388
Aspergillus in the Papanicolaou stain: morphology, fluorescence and diagnostic feasibility
Aspergillus species exhibit a distinct and clear fluorescence in Papanicolaou-stained cytological samples. The Papanicolaou (PAP) stain enhances the autofluorescence of cultured aspergilli and allows better cytological recognition of the fungus by fluorescence microscopy when it is not easily discerned from its surroundings by light microscopy. Morphological properties can be better distinguished and facilitate the differentiation of aspergillus organisms from other filamentous fungi. Neither light nor fluorescence microscopy, the cytological quality nor the presence of phagocytosed hyphae in alveolar macrophages allow distinction between infection and contamination with Aspergillus species. Only the presence of eosinophilic inflammation permits a tentative diagnosis of an Aspergillus infection. In conclusion, PAP fluorescence reduces the need for special stains, is superior to and quicker than other investigative techniques and enhances the sensitivity and specificity of cytological investigation when a rapid and reliable identification of Aspergillus is needed.     

A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.     

Decreased serum concentrations of nitric oxide metabolites among Chinese in an endemic area of chronic arsenic poisoning in inner Mongolia

Prolonged exposure to arsenic results in peripheral and cardiovascular manifestations, as does impaired production of endothelial nitric oxide (NO). In vitro studies have indicated that endothelial cells undergo damage by arsenic. However, no information has been available on the relationship between NO synthesis and chronic arsenic poisoning in humans. The present study was designed to reveal this question. The subjects were 33 habitants who continued to drink well water containing high concentrations of inorganic arsenic (mean value = 0.41 microg/ml) for about 18 years in Inner Mongolia, China, and 10 other people who lived in this area but exposed to minimal concentrations of arsenic (mean value = 0.02 microg/ml) were employed as controls. Mean blood concentration of total arsenic was six times higher in exposed subjects than controls; 42.1 vs. 7.3 ng/ml, p <.001. Mean serum concentration of nitrite/nitrate, stable metabolites of endogenous NO, was lower in arsenic-exposed subjects than in controls: 24.7 vs. 51.6 microM, p<.001. In total samples, an inverse correlation with serum nitrite/nitrate levels was strong for blood inorganic arsenic (r = -0.52, p <.001) and less strong for its metabolites, monomethyl arsenic (r = -0.45, p<.005) and dimethyl arsenic (r = -0.37, p<.05). Furthermore, serum nitrite/nitrate concentration was significantly correlated with nonprotein sulfhydryl level in whole blood (r = 0.58, p<.001). In an in vitro study, we demonstrated that inorganic arsenite or arsenate suppresses the activity of endothelial NO synthase in human umbilical vein endothelial cells. These results suggest that long-term exposure to arsenic by drinking well water possibly reduces NO production in endothelial cells, resulting in a decrease in reduced nitrite/nitrate concentrations. Peripheral vascular disorders caused by arsenic may be attributable in part to impairment of NO production in vivo.     

Fungal volatilization of trivalent and pentavalent arsenic under laboratory conditions

Production of volatile derivatives of arsenic was studied using pure cultures of different fungal strains under laboratory conditions. Arsenic was used in its trivalent and pentavalent forms to evaluate the effect of arsenic valency on its biovolatilization. The average amount of volatilized arsenic for all fungal strains ranged from 0.026 mg to 0.257 mg and 0.024 mg to 0.191 mg of trivalent and pentavalent arsenic, respectively. These results show that approximately 23% of arsenic was volatilized from all culture media originally enriched with approximately 4 and 17 mg L(-1) of arsenic in trivalent form. The average amount of biovolatilized arsenic from culture media originally enriched with 4 and 17 mg L(-1) of arsenic in pentavalent form was 24% and 16%, respectively. The order of ability of arsenic biovolatilization is Neosartorya fischeri > Aspergillus clavatus > Aspergillus niger. Toxicity and fungal resistance to trivalent and pentavalent arsenic were also evaluated based on radial growth and biomass weight.     

Fermentation extract effects on the morphology and metabolism of the rumen fungus Neocallimastix frontalis EB188

The effects of Aspergillus oryzae fermentation extract, Amaferm, on the rumen fungus Neocallimastix frontalis EB188 were studied. The secretion of cellulase was increased by 67% and rhyzoid development was increased 3.8-fold in the presence of extract. Strength of fungal response increased in a dose-dependent manner and demonstrated a positive correlation between cell surface area and enzyme secretion. Above certain concentrations of extract, however, the development of the fungus and enzyme secretions remained at control values or slightly diminished. Supernatant fluid appearance of the intracellular enzyme, malate dehydrogenase, paralleled the secretion of cellulase both in the presence and absence of extract. Ether solubilization of extract demonstrated that the active component(s) possessed a moderately polar value between 2.7 and 2.8. Thin layer chromatography separated extract into inert, inhibitory and intensely stimulating fractions. These results support the idea that by accelerating fungal growth and metabolism, Amaferm increases the rate (or extent) of fibre degradation caused by rumen fungi and that this, in turn, may contribute to enhanced animal performance.     

Uptake of reactive textile dyes by Aspergillus foetidus

An isolated fungus, Aspergillus foetidus was found to effectively decolorize media containing azo reactive dyes namely, Drimarene dyes. The extent of color removal was greater than 95% within 48 h of growth of the fungus. The entire color was found to be strongly bioadsorbed to the rapidly settling fungal biomass pellets without undergoing significant biotransformation. Our investigations reveal that the process of decolorization is concomitant with the exponential growth phase of the fungus and has requirement for a biodegradable substrate such as glucose. The fungus was also able to decolorize media containing mixture of dyes to an extent of 85% within 72 h of growth. Kinetic analyses of fungal decolorization indicate that the process is time dependent and follows first order kinetics with respect to initial concentration of dye. The rates of color uptake (k values) decrease to a significant extent with increasing initial concentrations of dye. The fungus was able to grow and decolorize media in the presence of 5 ppm of chromium and 1% sodium chloride. An alternate and cheaper carbon source such as starch supported the growth and decolorization process. These results suggest that dye uptake process mediated by A. foetidus has a potential for large-scale treatment of textile mill discharges.     

A protein kinase C-encoding gene, pkcA, is essential to the viability of the filamentous fungus Aspergillus nidulans

A protein kinase C (PKC)-encoding gene (pkcA) was isolated from the filamentous fungus Aspergillus nidulans. Although we attempted to isolate pkcA deletion mutants, we obtained only heterokaryons that had both DeltapkcA and pkcA(+) nuclei. Conidia produced by the heterokaryon germinated. The germ tubes, however, lysed frequently and no colony formation was observed, indicating that the pkcA gene is essential to the viability of A. nidulans. We constructed conditional mutants (alcA(p)-pkcA mutants) that expressed pkcA under the control of the alcA promoter (alcA(p)). Under alcA(p)-repressing conditions, their colonies were smaller than those of the wild-type strains and their hyphae lysed frequently. These phenotypes were not remedied under moderate- or high-osmolarity conditions; the growth defect deteriorated further under the latter. Under alcA(p)-inducing conditions, the alcA(p)-pkcA mutants also showed growth-sensitivity to cell wall destabilizing agents. These results indicate that pkcA plays an important role in the maintenance of cell integrity.     

Effect of selenite and tellurite on the morphological growth and toxin production of Aspergillus parasiticus var. globosus IMI 120920

Aspergillus parasiticus var. globosus IMI 120920 was able to grow in presence of different concentrations tested (0.052–4.0%) of sodium selenite or concentrations up to 2.0% potassium tellurite. Growth of the fungus was decreased greatly by the increase of metals concentrations. Dark colour colony and black reverse were formed in presence of tellurite while reddish gray to grayish red colony colour and brownish red to orange red reverse were formed in presence of selenite. The fungal biomass was slightly decreased at lower concentrations and highly inhibited at higher concentrations of selenite or tellurite. Selenite slightly stimulated aflatoxin formation at lower concentrations and highly inhibited it at higher concentrations. Aflatoxin production was decreased greatly by increasing tellurite concentrations. Obvious malformations were observed in the morphological features of the fungus in presence of different levels of selenite or tellurite. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aspterric acid and 6-hydroxymellein,inhibitors of pollen development in Arabidopsis thaliana,produced by Aspergillus terreus

Aspterric Acid, 6-Hydroxymellein, Arabidopsis thaliana, Aspergillus terreus Aspterric acid (1) and 6-hydroxymellein (2), inhibitors of pollen development in Arabidopsis thaliana, have been isolated from the fungus Aspergillus terreus. 1 and 2 inhibited the pollen development at concentrations of 38 and 52 microM, respectively. The microscopic examination of pollen development suggested that the inhibition by the treatment with 1 caused at meiosis and the inhibition by the treatment with 2 caused at microspore stage. 1 and 2 could be useful agents for the molecular investigation of anther and pollen development in higher plants.