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PubMed2004

Characterization of the Aspergillus parasiticus delta12-desaturase gene: a role for lipid metabolism in the Aspergillus-seed interaction.

In the mycotoxigenic oilseed pathogens Aspergillus flavus and Aspergillus parasiticus and the model filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives act as important developmental signals that affect asexual conidiospore, sexual ascospore and/or sclerotial development. To dissect the relationship between lipid metabolism and fungal development, an A. parasiticus Delta(12)-desaturase mutant that was unable to convert oleic acid to linoleic acid and was thus impaired in polyunsaturated fatty acid biosynthesis was generated. The Delta(12)-desaturase mutant demonstrates delayed spore germination, a twofold reduction in growth, a reduced level of conidiation and complete loss of sclerotial development, compared to the wild-type. Host colonization is impaired, as reflected by a decrease in conidial production on live peanut and corn seed by the mutant compared to the wild-type. Similarly, the previously isolated A. nidulans Delta(12)-desaturase mutant has reduced colonization capabilities compared to the wild-type. Therefore, desaturation mutants display a key requisite that affords a genetic solution to oilseed crop contamination by mycotoxigenic Aspergillus species: a reduction in the production of conidia, the infectious particle of the pathogenic aspergilli.

Arachis↗
PubMed1995

Differentiation of Aspergillus parasiticus from Aspergillus sojae by random amplification of polymorphic DNA.

Aspergillus parasiticus and Aspergillus sojae are two morphologically similar species belonging to the Aspergillus section flavi. A new method to distinguish the two species, the aflatoxin producer A. parasiticus and the koji mold A. sojae, was developed. Single primers with arbitrary sequences were used to generate random amplified polymorphic DNA (RAPD) markers from strains of these two species. Three decamers, OPA-04, OPB-10, and OPR-01, allowed adequate discrimination between strains of A. parasiticus and A. sojae in RAPD analyses. A. sojae was further separated into group I and group II with the three primers. On the other hand, A. parasiticus was divided into group A and group B when amplified with OPA-04 and OPR-10 primers. The previously misidentified stain CCRC 32423 and the misclassified strain CCRC 30227 were identified as Aspergillus flavus and A. sojae, respectively, on the basis of RAPD patterns and morphological characteristics. We suggest that the RAPD technique is a rapid and reliable tool to distinguish A. parasiticus from A. sojae.

Aspergillus↗
PubMed1994

Heterologous expression of the cytotoxin restriction in Aspergillus nidulans and Aspergillus niger.

The cDNA clone of restrictocin was placed under the control of the glucoamylase promoter from Aspergillus awamori and was transformed into Aspergillus nidulans and Aspergillus niger. Site-specific changes were introduced into cDNA constructs and these were transformed into A. nidulans. The secretion signal sequence was deleted from one form of the gene and three mutations introduced single amino acid substitutions into the protein. Culture conditions were optimized for maximum expression levels of restrictocin. The activities of the expressed proteins were characterized with an in vitro rabbit reticulocyte assay. Protein synthesis in this assay was inhibited 50% by 2.5 ng/ml wild-type restrictocin, 3.5 ng/ml E95G, 30 ng/ml E95C, and 600 ng/ml H136L. Toxic effects of restrictocin were observed in the A. nidulans expression system with reduced levels of cellular protein and messenger RNA upon induction of restrictocin expression as well as the formation of the alpha-fragment product of ribosomal RNA cleavage. Toxic effects were most highly pronounced in strains expressing restrictocin without the signal sequence, less so in strains expressing native restrictocin, and absent in strains expressing H136L restrictocin.

Allergens↗
PubMed1995

Effects of leader sequences upon the heterologous expression of restrictocin in Aspergillus nidulans and Aspergillus niger.

The effects of altered leader sequences on the secretion and localization of restrictiocin expression in Aspergillus nidulans and Aspergillus niger were investigated. The region encoding the leader sequence of the Aspergillus restrictus restrictocin (res) gene was altered and variants were expressed under the glucoamylase (glaA) promoter in A. nidulans and A. niger. The entire restrictocin leader sequence was replaced by the glaA leader sequence in one variant. In another, the signal sequence of restrictocin was replaced with the glaA signal, leaving a hybrid with the putative restrictocin pro region in place of the glaA pro region. The putative pro region was deleted from the restrictocin leader of a third variant. Toxic effects, such as reduced transcript levels and cellular lysis, were minimal when restrictocin was expressed with the native leader sequence, but became more pronounced as the leader sequence was varied. These toxic effects were inversely proportional to the level of restrictocin secreted. In all transformed strains, restrictocin secretion appeared at the periphery of colonies and was observed to occur at the tips of hyphae. Localization of restrictocin to differentiated structures (conidiophores), as occurs in A. restrictus, was observed only in transformed strains containing the complete restrictocin leader sequence.

Allergens↗
PubMed1978

Aspergillus fumigatus and Aspergillus niger in two potted ornamental plants, cactus (Epiphyllum truncatum) and clivia (Clivia miniata). Biological and epidemiological aspects.

The presence and growth of Aspergillus fumigatus and Aspergillus niger in the soil of ornamental plants have been demonstrated. The ecological conditions in the soil of such plants as influenced by temperature, humidity, desiccation, fertilization and ventilation obviously influence such fungal growth. The epidemiological significance of these findings is of interest with a view to the present efforts to control aspergillosis in the environment of susceptible persons. Observations of a preferential growth of certain Aspergillus species in the soil of defined plants under defined conditions raise problems of soil microbiology.

Aspergillosis↗
PubMedDate not supplied

Defective phagocyte Aspergillus killing associated with recurrent pulmonary Aspergillus infections.

An apparently healthy boy was suffering from recurrent Aspergillus infections. No classical conditions of immunodeficiency were found. Studies on the patient's phagocytic system revealed neutrophils and monocytes to function normally except in Aspergillus killing (microbicidal activity for bacteria and Candida was normal). Aspergillus killing mechanisms may be complex and peculiarly selective, possibly involving both oxygen-dependent and independent mechanisms.

Adolescent↗
PubMed1995

The Aspergillus parasiticus polyketide synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis.

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced by Aspergillus parasiticus and Aspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway in A. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipient A. parasiticus genome at a specific locus, designated pksA. Sequence analysis suggest that pksA is a homolog of the Aspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conserved beta-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of the pksA product. No beta-ketoacyl reductase and enoyl reductase domains were found, suggesting that pksA does not encode catalytic activities for processing beta-carbon similar to those required for long chain fatty acid synthesis. The pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose that pksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.

Aflatoxin B1↗
PubMed2007

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in Aspergillus flavus NRRL 3357 and Aspergillus parasiticus SRRC 143.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B(1) and B(2) biosynthesis, while A. parasiticus cultures had significantly increased B(1) and G(1) biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.

Aflatoxins↗
PubMed1985

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans.

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.

Arginine↗
PubMed2005

Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays.

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.

Aspergillus↗
PubMed2005

Detection of transcripts of the aflatoxin genes aflD, aflO, and aflP by reverse transcription-polymerase chain reaction allows differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and Aspergillus parasiticus.

The aim of this study was to test the suitability of the RT-PCR (reverse transcription-polymerase chain reaction) technique to differentiate aflatoxin-producing from aflatoxin-non-producing strains of Aspergillus flavus and Aspergillus parasiticus. Total RNAs of 13 strains grown under inducing yeast extract-sucrose (YES) and non-inducing yeast extract-peptone (YEP) media, respectively, were analyzed by using specific primers based on the conserved regions of nine structural genes (aflD, aflG, aflH, aflI, aflK, aflM, aflO, aflP, and aflQ) and two regulatory genes aflS and aflR of the aflatoxin B1 biosynthetic pathway. Transcription was confirmed by the expression of the beta-tubulin gene. The expression of the majority aflatoxin biosynthetic genes including aflR and aflS of all strains varied with regard to the aflatoxin-producing ability and the growth conditions. Nonetheless, we found that the expression profile of the three genes aflD, aflO, and aflP was consistently correlated with a strain's ability to produce aflatoxins or not in YES as well as the inability to produce aflatoxins in YEP. The devised RT-PCR profiling method reflects aflatoxin concentrations ranging from 0.1 to 60 microg/ml of the culture filtrates as determined by fluorescence HPLC. The results are discussed in relation to the suitability of RT-PCR as well as cDNA-based array techniques in diagnostic laboratory settings where individual isolates are being tested for potential toxin production to identify toxigenic isolates of Aspergillus species.

Aflatoxins↗
PubMed2002

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth.

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3',4'-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

Antifungal Agents↗
PubMed2005

Aflatoxin production in six peanut (Arachis hypogaea L.) genotypes infected with Aspergillus flavus and Aspergillus parasiticus, isolated from peanut production areas of Cordoba, Argentina.

Aflatoxin contamination is one of the main factors affecting peanut seed quality. One of the strategies to decrease the risk of peanut aflatoxin contamination is the use of genotypes with resistance to Aspergillus infection. This laboratory study reports the resistance to Aspergillus infection and aflatoxin contamination of six peanut genotypes inoculated with 21 Aspergillus isolates obtained from the peanut production region of Cordoba, Argentina. The resistance was investigated in the seed coat and cotyledons of three resistant genotypes (J11, PI 337394, and PI 337409) and three breeding lines (Manfredi 68, Colorado Irradiado, and Florman INTA) developed at the Instituto Nacional de Tecnologia Agropecuaria (INTA), Manfredi Experimental Station, Cordoba, Argentina. Resistance to fungal colonization and aflatoxin contamination was found to be associated with seed coat integrity in the PI 337394, PI 337409, and J11 genotypes, whereas the INTA breeding lines such as Colorado Irradiado showed a moderate resistance and the Manfredi 68 and Florman INTA genotypes the least resistance. Furthermore, another type of resistance associated with cotyledons was found only in the PI 337394 genotype.

Aflatoxins↗
PubMed1991

Analysis of Aspergillus nidulans conidial antigens and their prevalence in other Aspergillus species.

Aspergillus nidulans is an ascomycetous fungus that reproduces asexually by forming multicellular conidiophores and uninucleate spores called conidia. These elements constitute the main vehicle for the transmission of this and other pathogenic Aspergillus species and are the starting point of the different forms of aspergillosis. In order to use A. nidulans as a potential source of useful antigens for the immunodiagnosis of these diseases, we have examined the total protein composition of conidial extracts of this fungus by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in gels of different percent T. Injection of SDS-extracted conidial proteins into rabbits allowed us to raise a battery of polyclonal antibodies which have defined some important immunogenic polypeptides. Several of these immunogens were both present in mycelial extracts and recognized by antimycelium antibodies. Four of them, designated cdA, cdB, cdC, and cdE, were also found in conidial extracts of other pathogenic Aspergillus species. Only cdE was undetectable in cell extracts of the nonrelated species Fusarium culmorum and Phycomyces blakesleeanus.

Antigens, Fungal↗
PubMed1993

Typing of Aspergillus species and Aspergillus fumigatus isolates by interrepeat polymerase chain reaction.

Polymerase chain reaction amplification of repetitive DNA motifs allows discrimination of Aspergillus species. In a preliminary survey, the DNA fingerprints appear to be identical when isolates of Aspergillus fumigatus are compared. When a primer deduced from a prokaryotic repeat motif is used, Aspergillus fumigatus isolates originating from different patients or different anatomical locations can be typed individually.

Aspergillus↗
PubMed1983

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus.

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.

Aflatoxin B1↗
PubMed1999

Molecular and enzymic properties of recombinant 1, 2-alpha-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells.

For the construction of an overexpression system of the intracellular 1,2-alpha-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1, 2-alpha-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-alpha-d-mannopyranosyl alpha-D-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside or methyl-6-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type 'M8A') fastest, and 'M6C' {Manalpha1-3[Manalpha1-2Manalpha1-3(Manalpha1-6) Manalpha1-6]Manbeta1- 4GlcNAcbeta1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, epsilon, at 280 nm was determined as 91539 M-1.cm-1. Contents of the secondary structure (alpha-helix, beta-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, Tm, of the enzyme was 71 degrees C by differential scanning calorimetry. The calorimetric enthalpy, DeltaHcal, of the enzyme was calculated as 13.3 kJ.kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.

Amino Acid Sequence↗
PubMed1979

The effect of beta-glucuronidase and chitinase on the cell wall of Aspergillus niger and Aspergillus fumigatus.

The effects of beta-glucuronidase and chitinase have been tested on the hydrolysis of the cell walls of the economically important fungi, Aspergillus niger and Aspergillus fumigatus. The extent of wall hydrolysis was measured by assaying for total reducing sugars, N-acetyl sugars and protoplast production. Maximum reducing sugar release was attained after 40 min incubation, both with beta-glucuronidase supplemented with chitinase and beta-glucuronidase alone, whereas N-acetyl sugar release reached a maximum at 80 min incubation. beta-Glucuronidase was effective in releasing protoplasts from both species of Aspergillus. This release was enhanced by adding chitinase to the incubation medium at 0 and 20 min, but with addition at 60, 80 and 100 min increase in protoplast yield was much reduced. The results of re-incubation experiments with chitinase suggest that this enzyme may in some way be inhibited during the later stages of incubation. Pronase used in combination with beta-glucuronidase slightly enhanced protoplast release.

Aspergillus fumigatus↗