Identification of spore allergens from the indoor mould Aspergillus versicolor

Background:  Indoor mould growth and dampness are associated with respiratory health effects and allergies and several studies demonstrated that mainly Aspergillus versicolor and Penicillium expansum are responsible for indoor mould exposure. In contrast, commercialized test systems to diagnose allergic reactions to this mould species are not available. In this study, allergenic proteins from spores of the indoor relevant species A. versicolor and P. expansum should get detected and identified.
Methods:  We used two-dimensional (2D)-gel electrophoresis of spore proteins and immunoblotting with sera from patients participating in an epidemiologic study about indoor exposure of moulds and their influence on the development of allergies (ESTERSPEGA). Sera were screened for IgE antibodies specific for proteins from A. versicolor , A. fumigatus and P. expansum in one-dimensional blots and in 2D immunoblots. From the 2D gels, the corresponding spots were picked and identified by mass spectrometry.
Results:  More than 20 allergens from A. versicolor were identified; in particular, seven major allergens were selected, which were detected by more than 90% of the positive sera. The most abundant allergen was glyceraldehyde-3-phosphate dehydrogenase, followed by an unnamed protein, which displays a high homology to sobitol/xylose reductase. The other allergens were identified as catalase A, hypothetical protein AN6918.2, enolase, hypothetical protein AN0297.2 and a protein with homology to a fungal malate dehydrogenase.
Conclusions:  The results indicate an important role of spore proteins from A. versicolor for sensitization against indoor moulds and identification of the major allergens might enable species-specific diagnosis of allergic reactions.     

Gliotoxin production by clinical and environmental Aspergillus fumigatus strains

The mycotoxin gliotoxin is produced by fungi of the genus Aspergillus, including the important human pathogen Aspergillus fumigatus. Gliotoxin exerts a broad spectrum of immunosuppressive effects in vitro and is detectable in the sera of patients suffering from invasive aspergillosis. In order to correlate the pathogenic potential of A. fumigatus with the ability to produce gliotoxin and to investigate the taxonomic distribution of gliotoxin-producing Aspergillus strains among clinical isolates, a total of 158 Aspergillus isolates comprising four different species (A. fumigatus, n=100; A. terreus, n=27; A. niger, n=16; A. flavus, n=15) were collected from different medical centers (some originating from probable cases of aspergillosis) and from environmental samples in Germany and Austria. Remarkably, gliotoxin was detected in most culture filtrates of A. fumigatus of both clinical (98%) and environmental (96%) origin. The toxin was also detected, with decreasing frequency, in culture filtrates of A. niger (56%), A. terreus (37%), and A. flavus (13%). The highest gliotoxin concentrations were detected in A. fumigatus strains of clinical (max. 21.35 μg/ml, mean 5.75 μg/ml) and environmental (max. 26.25 μg/ml, mean 5.27 μg/ml) origin. Gliotoxin productivity of other Aspergillus species was significantly lower. Culture supernatants of A. fumigatus strains lacking gliotoxin production showed a significantly lower cytotoxicity on macrophage-like cells and T-cells in vitro. In contrast, lack of gliotoxin production in the other Aspergillus species tested had no significant influence on the cytotoxic effect of culture supernatant on these immune cells.     

Temperature effect in the production of multiple xylanases by Aspergillus fumigatus

This work has evaluated the temperature effect in the production of multiple xylanases by a locally isolated strain of Aspergillus fumigatus Fresenius. Three isoenzymes, identified as xylanases I, II, and III with apparent molecular weight of 45.7 KDa, 39.8 KDa and 18.2 KDa, respectively, were produced in cultures developed at 30 degrees C and at 42 degrees C. The pattern of distribution of xylanase activity among the three isoenzymes was greatly affected by the growth temperature: at 30 degrees C, the total xylanase activity was distributed homogeneously among the three enzymes, while at 42 degrees C, the total xylanase activity was mainly due to the fractions with the highest MW (I and II) and the xylanase III was a minor component.     

Effect of osmotic stress on glucose oxidase production and secretion by Aspergillus niger

A mycelium of Aspergillus niger was cultured under various osmotic stresses. When fungus was cultured under osmotic stress, total catalase activity increased with increasing concentration of NaCl, up 0.4 M , but glucose oxidase under this condition significantly decreased. Mycelial growth was repressed with increased media osmolarity. To release periplasmic glucose oxidase 72-h old mycelium was suspended on a concentrated solution of NaCl (0.4—2.8 M /l). The highest yield of GOD activity was obtained at a NaCl concentration of 1.2 M at pH 6.0, which improved the activity of this enzyme by about 2.1-fold in comparison with the control medium without this depressor.     

Characterization of the 41kDa allergen Asp v 13, a subtilisin-like serine protease from Aspergillus versicolor

Aspergillus versicolor is common on moldy building materials. Asp v 13, the principal allergen is produced by strains collected from across Canada. In this paper, we report a 1833bp Asp v 13 open reading frame predicted to encode a protein of 403 amino acids in length with three introns. A BLAST search of Asp v 13, a phylogenic tree calculation and alignment with its homologous proteins from other species indicated that Asp v 13 is a secretory, subtilisin-like serine protease widely distributed in Aspergillus species. His-tagged Asp v 13 was over-expressed in Escherichia coli and purified using Ni-NTA columns with a yield of 1mg/L. Based on immuno binding assay of recombinant protein both antibodies developed against the natural protein, and human sera IgE, the recombinant protein was similar to the natural form. Six IgE- and seven IgG-binding epitopes were also identified with selected human sera along the entire amino acid sequence of Asp v 13. Most residues binding these epitopes are exposed on the surface and correspond to charged regions of the molecule.     

Immunoglobulin G antibodies against environmental moulds in a Norwegian healthy population shows a bimodal distribution for Aspergillus versicolor

Immunoglobulin G (IgG) antibodies against moulds related to indoor dampness problems are used as biomarkers to indicate exposure. In the present study, we evaluated the frequency of mould exposure in an adult healthy population by examining levels of mould-specific IgG antibodies in Norwegian blood donors. Using enzyme-linked immunosorbent assay, 106 blood donor sera were analyzed for IgG antibodies to Aspergillus versicolor, Penicillium chrysogenum, Cladosporium herbarum, Stachybotrys chartarum and Fusarium oxysporum. The levels of specific IgG antibodies to P. chrysogenum, C. herbarum and S. chartarum correlated (r = 0.46-0.62). Responses to A. versicolor were considerably stronger than to the other moulds, and another 996 blood donor sera were analyzed for IgG antibodies to this mould. Women had significantly higher levels of specific IgG antibodies to A. versicolor than men. The concentration of A. versicolor-specific IgG antibodies showed a non-Gaussian, bimodal distribution profile, in which 12.5% were defined as positive to exposure. This suggests that significant mould exposure in a healthy population can be calculated from mean + 1SD. Western blotting analyses showed that antibody responses to A. versicolor were largely directed against carbohydrate antigens of unknown saccharides.     

L‐methioninase production by Aspergillus flavipes under solid‐state fermentation

Solid‐state fermentation was carried out for the production of extra‐cellular L‐methioninase by Aspergillus flavipes (Bain and Sart.) using nine agro‐industrial residues, namely wheat bran, rice bran, wheat flour, coconut seeds, cotton seeds, ground nut cake, lentil hulls, soya beans and chicken feathers. Chicken feathers were selected as solid substrate for L‐methioninase production by A. flavipes. The maximum L‐methioninase productivity (71.0 U/mg protein) and growth (11 mg protein/ml) of A. flavipes was obtained using alkali pretreated chicken feathers of 50% initial moisture content as substrate supplemented with D‐glucose (1.0% w/v) and L‐methionine (0.2% w/v). External supplementation of the fermentation medium with various vitamin sources has no overinductive effect on L‐methioninase biosynthesis. The partially purified A. flavipes L‐methioninase preparation showed highest activity (181 U/ml) at pH 8.0 with stability over a pH range (pH 6–8) for 2 h. L‐methioninase activity was increased by preincubation of the enzyme for 2 h with Co²⁺, Mn²⁺, Cu²⁺ and Mg²⁺ and strongly inhibited by the presence of EDTA, NaN₃, Li²⁺, Cd²⁺, DMSO and 2‐mercaptoethanol. The enzyme preparation has a broad substrate spectrum showing a higher affinity to deaminate L‐glycine, N ‐acetylglucosamine and glutamic acid, in addition to their proteolytic activity against bovine serum albumin, casein, gelatin and keratin. The partially purified enzyme was found to be glyco‐metalloproteinic in nature as concluded from the analytical and spectroscopic profiles of the enzyme preparation. The demethiolating activity of the enzyme was also visualized chromogenially. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Influence of growth conditions on the production of xylanolytic enzymes by Aspergillus flavus.

Investigations were carried out to optimize the culture conditions for the production of xylanase and beta-xylosidase by Aspergillus flavus, a filamentous fungus isolated from soil. The production of enzymes was tolerant to a wide range of initial culture pH values. Maximum xylanase (190 U/ml) and beta-xylosidase (35 U/ml) production was obtained when the strain was grown on mineral medium supplemented with 3% (w/v) corn cob powder as the carbon source. The enzymes had optimal activities at pH values between 5.5 and 6.0 and exhibited high activity and stability under alkaline conditions.     

Xylanolytic complex from Aspergillus giganteus: production and characterization

An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid Vogel medium containing xylan as carbon source, pH 6.5 to 7.0, at 25 degrees C and under shaking at 120 rpm during 84 h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar-cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50 degrees C and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T(50) of 2 h, 13 min and 1 min when it was incubated at 40, 50 and 60 degrees C, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg(+2), Cu(+2) and SDS at 10 mM. The analysis of the products from the oat spelts xylan hydrolysis through thin-layer chromatography indicated endoxylanase activity, lack of debranching enzymes and beta-xylosidase activity in assay conditions.     

Characterization of the 41 kDa allergen Asp v 13, a subtilisin-like serine protease from Aspergillus versicolor

Aspergillus versicolor is common on moldy building materials. Asp v 13, the principal allergen is produced by strains collected from across Canada. In this paper, we report a 1833 bp Asp v 13 open reading frame predicted to encode a protein of 403 amino acids in length with three introns. A BLAST search of Asp v 13, a phylogenic tree calculation and alignment with its homologous proteins from other species indicated that Asp v 13 is a secretory, subtilisin-like serine protease widely distributed in Aspergillus species. His-tagged Asp v 13 was over-expressed in Escherichia coli and purified using Ni-NTA columns with a yield of 1 mg/L. Based on immuno binding assay of recombinant protein both antibodies developed against the natural protein, and human sera IgE, the recombinant protein was similar to the natural form. Six IgE- and seven IgG-binding epitopes were also identified with selected human sera along the entire amino acid sequence of Asp v 13. Most residues binding these epitopes are exposed on the surface and correspond to charged regions of the molecule.     

Temperature and aflatoxin production by Aspergillus flavus and A. parasiticus strains from Nigerian groundnuts

The ability of four strains of Aspergillus parasiticus (IMI 301,001) and two strains of Aspergillus flavus (IMI 300,998) to produce aflatoxins on Nigerian groundnuts under varying conditions of temperature was studied. While all the A. parasiticus strains produced the four major aflatoxins (B1, B2, G1, G2), only aflatoxin B1, and B2 were produced by the A. flavus strains used. The optimum temperature for aflatoxin production by both the fungal species was 30 degrees C with no toxin production at 10 degrees C.     

Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence

Oxylipins comprise a family of oxygenated fatty acid-derived signaling molecules that initiate critical biological activities in animals, plants, and fungi. Mammalian oxylipins, including the prostaglandins (PGs), mediate many immune and inflammation responses in animals. PG production by pathogenic microbes is theorized to play a role in pathogenesis. We have genetically characterized three Aspergillus genes, ppoA, ppoB, and ppoC, encoding fatty acid oxygenases similar in sequence to specific mammalian prostaglandin synthases, the cyclooxygenases. Enzyme-linked immunosorbent assay analysis showed that production of PG species is decreased in both Aspergillus nidulans and A. fumigatus ppo mutants, implicating Ppo activity in generating PGs. The A. fumigatus triple-ppo-silenced mutant was hypervirulent in the invasive pulmonary aspergillosis murine model system and showed increased tolerance to H(2)O(2) stress relative to that of the wild type. We propose that Ppo products, PG, and/or other oxylipins may serve as activators of mammalian immune responses contributing to enhanced resistance to opportunistic fungi and as factors that modulate fungal development contributing to resistance to host defenses.     

Disseminated infection by Aspergillus terreus

A patient with acute nonlymphocytic leukemia who received chemotherapy developed lung nodules and later central nervous system symptoms consistent with disseminated aspergillosis. The diagnosis was made at open lung biopsy by culturing the organism and observing in tissue sections conidia borne laterally along the hyphae, a characteristic of the Aspergillus terreus-flavipes group. This is the first reported case of disseminated A. terreus infection in an immunocompromised host.     

HLA antigens in pityriasis versicolor

The distribution of 30 HLA antigens was studied in 48 patients with Pityriasis Versicolor and 134 controls. No significant deviations were found in the patient group after correction for the number of antigens tested.     

Aspergillus fumigatus complement inhibitor: production, characterization, and purification by hydrophobic interaction and thin-layer chromatography.

Aspergillus fumigatus has previously been shown to produce a soluble extracellular inhibitor of the alternative complement pathway, called Aspergillus complement inhibitor, or CI. We now report an efficient method for production of CI which relies on the fact that poorly conidiating cultures yielded CI activity with approximately sevenfold-higher potency than CI produced by conidiating cultures. CI from poorly conidiating cultures provided 50% inhibition of alternative pathway-mediated binding of 125I-labeled complement component C3 to cryptococcal blastoconidia at a mean concentration of 60 micrograms/ml. The ability of crude CI to inhibit the alternative complement pathway seemed to be independent of intact protein or polysaccharide structure, as evidenced by resistance of inhibitory activity to digestion by proteases, including subtilisin, alpha-chymotrypsin, papain, and pepsin as well as endoglycosidases F and H. Separation of the active inhibitory component of CI from contaminating materials contained in crude CI preparations was achieved by using Phenylsuperose hydrophobic interaction chromatography in a fast protein liquid chromatography system. The active material proved to be extremely hydrophobic, desorbing from the column only during elution with ethanol; it contained only 15% protein and 5% polysaccharide. Furthermore, results from preparative thin-layer chromatography indicated that lipids which comigrated with phosphatidylserine/phosphatidylinositol and phosphatidylethanolamine possessed significant complement-inhibitory activity. Taken together, these data suggested that phospholipids from A. fumigatus contributed to the functional activity of CI.     

Alcohol production from starch by mixed cultures of Aspergillus awamori and immobilized Saccharomyces cerevisiae at different agitation speeds

The influence of different agitation speeds on alcoholic fermentation by free and immobilized cells of Saccharomyces cerevisiae in a co-culture with free cells of Aspergillus awamori was investigated. Starch hydrolysis and glucose, glucoamylase and alpha-amylase accumulation in the fermentation medium was affected by agitation speed. Maximum amounts of the enzymes were obtained at 150-200 rpm after 72 h where, maximum growth and glucose accumulations were noticed at 200-300 rpm. Alcohol production was stimulated by low agitation speed (50 rpm) and decreased at higher speeds. The results also showed that the amount of produced alcohol was affected by the time of yeast inoculation. When the inoculation of yeast was carried out after the growth of fungi for 72 h, the amounts of produced alcohol increased by 84, 75, 89 and 68% at 50, 100, 150 and 200 rpm, respectively than that produced when the two organisms were inoculated together at the beginning of the fermentation process. A batch culture of the two organisms produced about 2% (v/v) alcohol from 12% (w/v) corn starch in 72 h at 50 rpm. On the other hand, the immobilized yeast and suspended A. awamori produced more alcohol reaching 3.7% (v/v) at 200 rpm under the same cultivation conditions. The fermentation process was less affected by alginate and cell concentrations of the immobilized yeast. Repeated batch fermentation by co-culture of A. awamori and immobilized yeast cells were successfully used for 12 times without a significant loss in alcohol production.     

Cerebral Aspergillosis Caused by Aspergillus granulosus

Disseminated disease by Aspergillus granulosus has been reported only once previously in a cardiac transplant recipient. We report a fatal central nervous system infection in a lung transplant recipient. Key features of this species in the section Usti include growth at 37°C and large, randomly spaced aggregates of variably shaped Hülle cells.     

Sclerosing mediastinitis caused by Aspergillus terreus

A case of Aspergillus terreus causing sclerosing mediastinitis which presented with symptoms of cardiorespiratory compromise and compressive myelopathy is described. The diagnosis was established by culturing and isolating the fungus in pure culture from the tissue and was also confirmed by demonstration of sepcific precipitating antibodies against Aspergillus terreus in patient's serum.