Production of xylanases byAspergillus fumigatus andAspergillus oryzae on xylan-based media

Aspergillus fumigatus andA. oryzae were cultivated in laboratory fermenters on media containing xylan as the main carbon source.A. fumigatus produced xylanase on unsubstituted, insoluble beech xylan but growth and enzyme production on soluble xylo-oligosaccharides from the steaming of hardwood were poor due to the presence of inhibitors. An essential prerequisite for good xylanase production byA. fumigatus was decrease in the pH of the cultivation below 3.0 At higher pH values, the production of proteolytic enzymes caused degradation of the xylanase activity already produced.A. oryzae produced rather less xylanase activity thanA. fumigatus on the beech xylan medium but, after adaptation, was capable of efficient enzyme production on the steamed substrate.M.J. Bailey and L. Viikari are with the VTT, Biotechnical Laboratory, PO Box 202, SF-02151 Espoo, Finland     

Penicillium andAspergillus species in the habitats of allergy patients in the Tulsa, Oklahoma area

Penicillium andAspergillus have been recognized as important aeroallergens for more than 30 years, and are especially significant in indoor environments. There are over 400 species ofPenicillium andAspergillus combined, but there is little information on which species occur most frequently in the environment, or if each exhibits unique allergenic properties. A preliminary study showed no overlap between those species isolated from an outdoor site in Tulsa, Oklahoma and the species used in immunotherapy at allergy clinics in the Tulsa area. Pursuing this line of research, air samples were collected as three seasonal samples (over a 6 month period) in the homes or offices of ten allergy patients known to be allergic toPenicillium and/orAspergillus. Twenty three species ofPenicillium and 12 species ofAspergillus were identified from these samples through isolation, macroscopic, and microscopic examination.Penicillium corylophilum, P. glabrum, Aspergillus niger, andA. flavipes were the most abundant species isolated, supporting the data obtained in a preliminary study. At least in the Tulsa area, it appears that atopic patients are being tested and treated with extracts ofPenicillium andAspergillus species that are either not present or not abundant in the local indoor or outdoor environments. Additional research is necessary to determine if the environmental isolates share allergens with those species used in immunotherapy.     

Clinical and experimental mycotic corneal ulcer caused by Aspergillus fumigatus and the effect of oral ketoconazole in the treatment

Aspergillus fumigatus was isolated from a case of keratomycosis. The patient, a 12-year-old boy presented with large corneal ulcer with hypopyon. The direct microscopic examination of scrapings revealed hyaline, septate mycelium. In vitro some antimycotics (amphotericin B,5-fluorocytosine, oxiconazole, amorolfine and ketoconazole) were tested against A. fumigatus by agar dilution method. Ketoconazole with minimum inhibitory concentration of 30 g/ml after 11 days of incubation was most effective against A. fumigatus. Experimental corneal ulcer was produced by injecting intralamellary spore suspension (2.5×10⁶ c.f.u.) into the right eyes of previously immunosuppressed albino and black wild types of rabbits. The extent of ocular infection was graded up to 32 days. Histopathologic examination showed infiltration and large destruction of corneal stroma. Oral ketoconazole therapy exhibited partial response followed by relapse. The black type of rabbit appeared more suitable as an animal model for mycotic keratitis.This paper was presented at the X^th congress of the International Society for Human and Animal Mycology at Barcelona, Spain from June 27 to July 1, 1988.     

Cloning and expression of pkaC and pkaR,the genes encoding the cAMP-dependent protein kinase of Aspergillus fumigatus

This report describes the cloning and expression of both subunits of PKA in the opportunistic fungal pathogen Aspergillus fumigatus. The predicted translation product of the regulatory subunit, pkaR, is defined as a type II regulatory subunit. The gene encoding the A. fumigatus catalytic subunit, pkaC, contains the conserved kinase and activation domains that are characteristic of PkaC proteins. Both subunit mRNAs are expressed throughout the asexual life cycle of A. fumigatus. Message levels of pkaR and pkaC are higher during co-cultivation with alveolar epithelial cells than during culture alone.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effects of some groundnut packaging methods and protection with Ocimum and Syzygium powders on kernel infection by fungi

Powders from the leaves of Ocimum gratissimum and cloves of Syzygium aromaticum were used as protectants at 3% (w/w) in combination with various packaging methods to store 3.5 kg groundnut kernel samples (9.3% moisture) artificially inoculated with Aspergillus parasiticus. Phostoxin-protected and unprotected samples were the controls. Packaging was accomplished with (i) Jute bags; JB (ii) Interlaced polypropylene bags; IPPB (iii) Polyethylene bags; PB (iv) PB inserted into IPPB and (v) PB inserted into JB. Selected treatments were repeated concurrently with naturally infected kernels (6.6% moisture). With 9.3% moisture kernels, there was a highly significant protectant, packaging method, and protectant X packaging method effect on protection of kernels from fungal infection at 2, 4, and 6 months. Packaging with JB and IPPB with or without plant powders gave 100% protection against fungi but insect infestation was prevented only when the Syzygium powder was used. When PB was used either singly or in combination with JB and IPPB, 100% protection from fungi was achieved up to 2 months with the Ocimum and up to 4 months with the Syzygium powder. The phostoxin treatment also gave 100% protection with JB and IPPB packaging but was ineffective with PB packaging. Kernels packaged with PB without the powders were extensively mouldy. Kernels with natural mycoflora (6.6% moisture) were free from fungi at 6 months regardless of the protectant and packaging used. In further tests, the Syzygium powder, at 3% and in combination with JB-packaging, effectively suppressed cross infection of healthy kernels (12% moisture) by fungi from diseased kernels when both kernel types occurred in the same lot. At 18.5% kernel moisture and with identical packaging, the Syzygium powder at 3%, was not as effective. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [→ 3)-β-D-Galf-(1 → 5)-β-D-Galf-(1 →] _n → mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [→ 5)-β-D-Galf-(1 →] _n → mannan core. The mannan cores have also been investigated, and are constituted by a (1 → 6)-α-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 → 2) linked α-mannopyranoses. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Survey of Vietnamese coffee beans for the presence of ochratoxigenic Aspergilli

Vietnamese coffee beans were investigated for the presence of ochratoxigenic Aspergilli. Ninety-three percent of the coffee samples studied were positive for A. niger. No other ochratoxigenic species were present. HPLC analysis determined that 8.7% of the A. niger strains were positive for ochratoxin A (OA) production. There was no significant difference in the level of contamination or incidence of toxigenic strains in samples that had been rejected by manual sorting and those that were destined for human consumption. No OA-producing fungi were uncovered in a fresh coffee bean sample analysed, suggesting that the OA problem most likely occurs post-harvest.     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Double infection of tobacco plants by two complementing octopine T-region mutants of Agrobacterium tumefaciens

The molecular basis of complementation by a mixture of two different types of octopine T-region mutants (LBA4060 and LBA4210) was studied. Six randomly chosen cellular clones derived from a tumor obtained after mixed infection were analyzed for their T-DNA content via Southern blot hybridization. The clones appeared to contain T-DNA that originated from each of both mutants, indicating that they developed from doubly infected single cells. Genetic complementation, therefore, might explain at least in part the observed complementation phenomenon. However, complementation as a result of cross-feeding between separately transformed cells could not be excluded. Following protoplast isolation, small aggregates might have formed that developed into the clones analyzed.     

Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.     

Invasive filamentous fungal infections in allogeneic hematopoietic stem cell transplant recipients after recovery from neutropenia: Clinical, radiologic, and pathologic characteristics

Invasive filamentous fungal infection (IFFI) is an important cause of mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We reviewed 22 consecutive cases of IFFI in allogeneic HSCT recipients at Roswell Park Cancer Institute. IFFI was diagnosed after neutrophil recovery in 21 patients (95%). All had received corticosteroids within 1 month prior to IFFI diagnosis. Fourteen (64%) presented with dyspnea, and only 7 (32%) were febrile. Aspergillus species were isolated in 18 (82%) cases. Thirty day mortality after IFFI diagnosis was associated with a higher mean daily dose of corticosteroids (P = 0.02) and receiving OKT3 (P = 0.01) within 1 month prior to IFFI diagnosis and serum creatinine >2 mg/dl at the time of diagnosis (P = 0.004). Histopathologic material from biopsy or autopsy was available in 15 patients (68%). In 8 (53%), the predominant lung histopathology was an acellular coagulative necrosis and hyphal angioinvasion was observed in some of these cases. These findings have generally been observed in neutropenic patients but not in non-neutropenic HSCT recipients. The predominance of coagulative necrosis in our series may reflect the high doses of corticosteroids used to treat graft-versus-host disease (GVHD), which may have disabled leukocyte trafficking and hyphal killing.     

Effect of culture temperature on the heterologous expression of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> versatile peroxidase in <Emphasis Type="Italic">Aspergillus</Emphasis> hosts

Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 °C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L^-1 was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.     

C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance ¹³C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The -alanine level increases further upon incubation with the non-permissive substrate -glucose. -Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with -glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on -fructose and -glutamate, alanine also accumulates within 3 h upon transfer to -glucose.     

Atkinsiella infection in the rotiferBrachionus plicatilis

Atkinsiella infection in the rotiferBrachionus plicatilis has not hitherto been reported. The first known case of this infection is described herein. The fungal growth in vitro was inhibited by 0.05µg/ml of malachite green or 15.6 ppm of formalin. It was experimentally demonstrated that the fungus is pathogenic to the swimming crab,Portunus trituberculatus in the zoeal stage.     

Stress factor-induced changes in the activity of antioxidant protective mechanisms in the wild type strain of <Emphasis Type="Italic">Neurospora crassa</Emphasis> and in its photoreceptor complex mutants

The effect of stress factors (changes in oxygen content, temperature, and illumination) on superoxide dismutase (SOD) and catalase activity, as well as on the content of thiol and disulfide groups, in low-molecular-weight compounds and proteins of Neurospora crassa mycelium was studied in the wild type strain and white collar-(wc-1) and white collar-2 (wc-2) mutants. Environmental stress factors induced the activation of both SOD and catalase, as well as an increase in the thiol level in the wild type strain of Neurospora crassa. In the wc-1 and wc-2 mutants, an increase in catalase activity and in the total thiol level was revealed; however, activation of superoxide dismutase was not observed. A decrease in the formation of disulfide bonds in the proteins of wc-1 and wc-2 mutants (as compared with the wild type strain) was recorded. These results indicate disrupted transduction of stress factor signals that promotes reactive oxygen species (ROS) formation in the WCC mutants.     

Production of extracellular enzymes and aflatoxins in solid substrate fermentation with aflatoxigenic<Emphasis Type="Italic">Aspergillus</Emphasis> spp.

AflatoxigenicAspergillus flavus andAspergillus parasiticus were subjected to solid substrate fermentation process for 6 days to determine the formation of aflatoxins and production of extracellular enzymes (amyloglucosidase, cellulase, invertase and proteinase). Both organisms produced enzymes which generally increased with fermentation.Aspergillus flavus produced four enzymes whereasA. parasiticus produced three with no proteinase activity.Aspergillus parasiticus produced aflatoxins B₁, B₂ and G₁ but no G₂ andA. flavus produced aflatoxins B₁ and B₂. Invertase showed the highest activity withA. parasiticus and that corresponded with the highest total toxin produced. The enzyme activities were higher withA. parasiticus thanA. flavus although total toxins produced byA. parasiticus were lower than total toxins produced byA. flavus under the same environmental conditions.     

<Emphasis Type="Italic">Aspergillus vadensis</Emphasis>, a new species of the group of black Aspergilli

A strain from the group of black Aspergilli was analysed in detail to determine the species to which it belongs. A detailed analysis of morphology, RFLP patterns and metabolite profiles was carried out. In addition, a phylogenetic tree was constructed for the black Aspergilli using the ITS and the -tubulin sequences of the individual strains. The new species differs by its poor growth on glycerol and galacturonate and its unique extrolite profile consisting of aurasperone B, nigragillin, asperazine and kotanins. RFLP analysis using three genes as probes also resulted in a unique pattern. These data indicate that the strain was closely related but not identical to Aspergillus foetidus, Aspergillus niger and Aspergillus tubingensis. It was therefore designated as a novel species and named Aspergillus vadensis.     

Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with nodulation mutants

Summary Symbiotic mutants of Rhizobium meliloti were isolated following Tn5 mutagenesis. Besides four nodulation mutants (Nod^-) unable to induce nodule formation on alfalfa, five infection mutants (Inf^-), which induce the formation of root nodules without detectable infection threads or bacteroids, were obtained. The Inf^- mutants were subdivided into two classes. One class contains mutants which fail to synthesize acidic exopolysaccharide (EPS^-). The other class is comprised of mutants which produce excess amounts of acidic exopolysaccharide (EPS^*). ¹³C nuclear magnetic resonance spectroscopy of the exopolysaccharide isolated from one of the latter type of Inf^- mutant, 101.45, revealed that the side chain of the repeating octosaccharide unit lacks the terminal pyruvate residue. Complementing cosmids were isolated for all Inf^- mutants. In the case of the Inf^- EPS^- mutants the complementing cosmids contain DNA segments which overlap and are part of megaplasmid 2. For two mutants the mutations were found to map on a 7.8 kb EcoRI fragment. In the case of the Inf^- EPS^* mutants the complementing cosmids carry chromosomal DNA. The mutations of two Inf^- EPS^* mutants were localized on a 6.4 kb EcoRI fragment. Coinoculation of alfalfa plants with Nod^- and Inf^- EPS^- mutants resulted in effective symbiosis. The nodules appeared wild type and fixed nitrogen. In constrast, coinoculations with Nod^- mutants and the Inf^- EPS^* mutant 101.45 did not result in the formation of effective nodules.