Fungi Aspergillus Parasiticus Research Articles

Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO 2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO 2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO 2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.

Catalytic Relationships between Type I and Type II Iterative Polyketide Synthases: The Aspergillus parasiticus Norsolorinic Acid Synthase

Norsolorinic acid synthase (NSAS) is a type I iterative polyketide synthase that occurs in the filamentous fungus Aspergillus parasiticus. PCR was used to clone fragments of NSAS corresponding to the acyl carrier protein (ACP), acyl transferase (AT) and beta-ketoacyl-ACP synthase (KS) catalytic domains. Expression of these gene fragments in Escherichia coli led to the production of soluble ACP and AT proteins. Coexpression of ACP with E. coli holo-ACP synthase (ACPS) let to production of NSAS holo-ACP, which could also be formed in vitro by using Streptomyces coelicolor ACPS. Analysis by mass spectrometry showed that, as with other type I carrier proteins, self-malonylation is not observed in the presence of malonyl CoA alone. However, the NSAS holo-ACP serves as substrate for S. coelicolor MCAT, S. coelicolor actinorhodin holo-ACP and NSAS AT domain-catalysed malonate transfer from malonyl CoA. The AT domain could transfer malonate from malonyl CoA to NSAS holo-ACP, but not hexanoate or acetate from either the cognate CoA or FAS ACP species to NSAS holo-ACP. The NSAS holo-ACP was also active in actinorhodin minimal PKS assays, but only in the presence of exogenous malonyl transferases.

Biocontrol of Aflatoxins B1,B2,G1,G2, and Fumonisin B1 with 6,7-Dimethoxycoumarin, a Phytoalexin from Citrus sinensis

Phytoalexins (stress-induced compounds) from Citrus sinensis cultivar Valencia were screened for antifungal and anti-mycotoxic activity against a test organism (Cladosporium cladosporoides) and mycotoxin-producing fungi Fusarium verticillioides and Aspergillus parasiticus. The active compound, a member of the coumarin family of compounds, has antifungal and antimycotoxic activities and was chemically identified. High-performance liquid chromatography results indicated that Valencia oranges contain a trace amount (0.36 μg/g) of scoparone in untreated fruit, but concentrations increased in UV-irradiated fruit (15.2 μg/g). Infection with Penicillium digitatum, a natural spoilage mold of citrus fruit, caused a 35.51-μg/g increase in the phytoalexin. UV absorption, infrared absorption, and 1H nuclear magnetic resonance spectroscopy revealed that this phytoalexin is identical to 6,7-dimethoxycoumarin. This is the first report indicating that the stress-induced compound, 6,7-dimethoxycoumarin, isolated from P. digitatum–infected Valencia fruit confers resistance against the mycotoxigenic fungi A. parasiticus and F. verticillioides and causes a reduction in production of fumonisin B1 and aflatoxins G1,G2,B1, and B2.

Distribution and sub-cellular localization of the aflatoxin enzyme versicolorin B synthase in time-fractionated colonies of Aspergillus parasiticus.

Aflatoxins are highly toxic and carcinogenic fungal secondary metabolites. At least 18 enzyme activities are required for aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus. One of these enzymes, versicolorin B synthase (VBS), catalyzes bisfuran ring closure in versiconal hemiacetal (a reaction near the middle of the pathway) to form versicolorin B. This reaction is required for the subsequent activation to aflatoxin B1-8,9 epoxide, a highly reactive and toxic aflatoxin metabolite, and is important for aflatoxin toxicity. We analyzed the localization of VBS in the aflatoxin-producing strain A. parasiticus SU-1 grown on solid media using a colony fractionation technique developed previously. A highly specific polyclonal antibody, raised against a maltose-binding protein-VBS fusion protein synthesized in Escherichia coli, was used to detect VBS in SU-1 grown on a rich solid medium via immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold transmission electron microscopy (TEM). VBS was detected in both vegetative hyphae and in asexual developmental structures, called conidiophores. Western blot and CLSM analyses demonstrated the highest abundance of VBS in colony fraction S2 consisting of cells that had grown for 24-48 h; this fraction also contained the highest levels of newly developed conidiophores and the highest abundance of aflatoxin B1, consistent with VBS abundance. At the subcellular level, CLSM and TEM detected VBS distributed throughout the cytoplasm and concentrated in ring-like structures surrounding nuclei. It is uncertain whether enzymatically active VBS is present in either or both locations.

A Novel cAMP-response Element, CRE1, Modulates Expression of nor-1 in Aspergillus parasiticus

The level of aflatoxin accumulation in the filamentous fungus Aspergillus parasiticus is modulated by a variety of environmental cues. The presence of glucose (a preferred carbon source) in liquid and solid glucose minimal salts (GMS) growth media strongly stimulated aflatoxin accumulation. Peptone (a non-preferred carbon source) in peptone minimal salts (PMS) media stimulated only low levels of aflatoxin accumulation. Glucose stimulated transcription of the aflatoxin structural genes ver-1 and nor-1 to similar intermediate levels in liquid GMS, while on solid media, ver-1 transcription was stimulated to 20-fold higher levels than nor-1. PMS liquid and solid media stimulated very low or non-detectable levels of transcription of both genes. Electrophoretic mobility shift analysis using a nor-1 promoter fragment (norR) and A. parasiticus cell protein extracts revealed specific DNA-protein complexes of different mobility on GMS and PMS solid and liquid media. An imperfect cAMP-response element, CRE1, was identified in norR that mediated formation of the specific DNA-protein complexes. Mutation in CRE1 or AflR1 (AflR cis-acting site) caused up to a 3-fold decrease in cAMP-mediated stimulation of nor-1 promoter activity on GMS agar. South-Western blot analysis identified a 32-kDa protein that specifically bound to norR. p32 could be co-immunoprecipitated by anti-AflR antibody and co-purified with an AflR-maltose-binding protein fusion demonstrating a physical interaction between AflR and p32 in vitro. We hypothesize that p32 assists AflR in binding to the nor-1 promoter, thereby modulating nor-1 gene expression in response to environmental cues.

Controlling Aflatoxin and Fumonisin in Maize by Crop Management

Maize is a vital food and feed grain worldwide. Aflatoxin and fumonisin, mycotoxins produced primarily by the fungi Aspergillus flavus and Aspergillus parasiticus Speare, and Fusarium moniliforme J. Sheld, respectively, are very potent carcinogens in both humans and livestock and can readily contaminate maize grain in the field and in storage. Stress on developing maize, particularly during reproductive growth, facilitates infection by the fungi, production of mycotoxins and contamination of the grain. Drought, excessive heat, inadequate plant nutrition, insect feeding on developing kernels, weeds, excessive plant populations, and other plant diseases can produce plant stress and facilitate the infection of maize grain by mycotoxin producing fungi. Timely planting of adapted hybrids, proper plant nutrition, irrigation, and insect control either by insecticides or the use of transgenic hybrids all assist in curbing mycotoxin contamination. Production practices that produce high yields are basically the sam...

Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus.

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-beta-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.

Efficacy and fumigation characteristics of ozone in stored maize

This study evaluated the efficacy of ozone as a fumigant to disinfest stored maize. Treatment of 8.9 tonnes (350 bu) of maize with 50 ppm ozone for 3 d resulted in 92–100% mortality of adult red flour beetle, Tribolium castaneum (Herbst), adult maize weevil, Sitophilus zeamais (Motsch.), and larval Indian meal moth, Plodia interpunctella (Hübner) and reduced by 63% the contamination level of the fungus Aspergillus parasiticus Speare on the kernel surface. Ozone fumigation of maize had two distinct phases. Phase 1 was characterized by rapid degradation of the ozone and slow movement through the grain. In Phase 2, the ozone flowed freely through the grain with little degradation and occurred once the molecular sites responsible for ozone degradation became saturated. The rate of saturation depended on the velocity of the ozone/air stream. The optimum apparent velocity for deep penetration of ozone into the grain mass was 0.03 m/s, a velocity that is achievable in typical storage structures with current fans and motors. At this velocity 85% of the ozone penetrated 2.7 m into the column of grain in 0.8 d during Phase 1 and within 5 d a stable degradation rate of 1 ppm/0.3 m was achieved. Optimum velocity for Phase 2 was 0.02 m/s. At this velocity, 90% of the ozone dose penetrated 2.7 m in less than 0.5 d. These data demonstrate the potential usefulness of using ozone in managing stored maize and possibly other grains.

Characterization of microsatellite loci in the aflatoxigenic fungi Aspergillus flavus and Aspergillus parasiticus

Characterization of microsatellite loci in the aflatoxigenic fungi Aspergillus flavus and Aspergillus parasiticus

Effects of volatile aldehydes from Aspergillus-resistant varieties of corn on Aspergillus parasiticus growth and aflatoxin biosynthesis

The fungi Aspergillus flavus and Aspergillus parasiticus produce a potent class of hepatocarcinogens known as aflatoxins. Corn-derived volatile compounds have been previously found to affect growth and aflatoxin production in A. flavus. In this study, the effects on A. parasiticus of three corn-derived volatile compounds, n-decyl aldehyde, hexanal and octanal, were measured. These three compounds were previously found to be variably expressed in five Aspergillus-resistant maize strains and three susceptible strains. In this study, A. parasiticus radial growth was restricted least by n-decyl aldehyde and most by octanal. Treatments of 100 μl of both hexanal and octanal were found to completely inhibit radial growth of the fungus using an agar plate assay method. While the volatile compound n-decyl aldehyde had less of an effect on radial growth than the other volatiles, the n-decyl aldehyde treated colonies had a predominance of uniquely aerial hyphae. These colony structures were found to have more complex hyphae and significantly fewer conidiophores than the control and other aldehyde treatments. Furthermore, aflatoxin production by the fungus was reduced by n-decyl aldehyde and hexanal, but was stimulated by octanal. The results presented here indicate that all three volatile compounds reduce radial growth but only n-decyl aldehyde significantly inhibits aflatoxin biosynthesis in A. parasiticus.

Possible Synergistic Effect of Nisin and Propionic Acid on the Growth of the Mycotoxigenic Fungi Aspergillus parasiticus, Aspergillus ochraceus, and Fusarium moniliforme

The effects of nisin and propionic acid (PA) on aflatoxin production and on mycelial growth and spore germination of the mycotoxigenic fungi Aspergillus parasiticus, A. ochraceus, and Fusarium moniliforme were investigated. The growth of A. ochraceus was completely inhibited on media containing PA with nisin in concentrations of 0.05% PA with 1,000 ppm nisin, and 0.1% PA with 500 or 1,000 ppm nisin. The growth of both F. moniliforme and A. parasiticus was completely inhibited by PA with nisin at a concentration of 0.1% PA with 1,000 ppm nisin. Nisin alone caused a significant increase in mycelial growth when applied to A. ochraceus at 500 or 1,000 ppm and when applied to A. parasiticus at 1,000 ppm. Spore germination of A. ochraceus was completely inhibited on media containing 0.1% PA with 500 or 1,000 ppm nisin. Spores of F. moniliforme failed to germinate in 0.05% PA with 500 or 1,000 ppm nisin, whereas spores of A. parasiticus did not germinate on media containing 0.1% PA with 1,000 ppm nisin. For all three fungi tested, the inhibitory effect on mycelial growth was found to be fungistatic rather than fungicidal. The combined treatment of PA with nisin produced better fungistatic activity than treatment involving either material alone. Nisin, applied alone, did not stimulate aflatoxin production (expressed by μg toxin/mg mycelium), but the combined treatment at certain concentrations was inhibitory to aflatoxin B1 or G1. The production of aflatoxin G1, but not of B1, was stimulated in 0.05% PA with 1,000 ppm nisin and on media containing 0.1% PA with 100 ppm nisin. Nisin is currently applied in foods to prevent spoilage induced by bacteria but not by mold. The results of the present study indicate that a combined treatment of nisin in small concentrations of PA might be useful in preventing mold damage in certain foods and stored grain.

Sequoiatones A and B: Novel Antitumor Metabolites Isolated from a Redwood Endophyte.

Sequoiatones A and B were isolated from the fungus Aspergillus parasiticus, an endophytic fungus of the coast redwood, Sequoia sempervirens. The compounds were isolated from the methanol extract of the mycelial mat of the fungus when grown in liquid culture for 21 days. The compounds were isolated because of their brine shrimp lethality, which served as an excellent guide for chromatographic purification. Full details of the isolation and characterization of sequoiatones A and B are provided herein, along with the test results from the NCI human tumor 60 cell-line screen.

The effects of selected cotton-leaf volatiles on growth, development and aflatoxin production of Aspergillus parasiticus

The fungi Aspergillus flavus and Aspergillus parasiticus produce the hepatocarcinogenic, secondary metabolites, aflatoxins, in cottonseed, corn, peanuts and treenuts. Results have shown that aflatoxigenic strains of A. flavus and A. parasiticus grown in the presence of specific cotton-leaf volatiles exhibit alterations in aflatoxin production accompanied by variations in growth of the fungi. In this study, two alcohols (3-methyl-1-butanol (3-MB) and nonanol) and two terpenes (camphene and limonene) were chosen as representative cotton-leaf volatiles based on the effects they had on fungal growth and/or aflatoxin production in previous investigations. The morphological effects of volatile exposure were examined in correlation with fungal growth and aflatoxin production. 3-MB-treated samples exhibited a decrease in fungal radial growth which was directly proportional to the volatile dosage. Additionally, 3-MB treatment resulted in loss of mycelial pigmentation and a decrease in sporulation. Limonene and camphene-treated samples yielded negligible differences in radial growth and morphology when compared to unexposed controls. In addition to radial growth inhibition, samples grown in the presence of nonanol demonstrated uniquely aerial hyphae. In comparison to an unexposed control, aflatoxin production increased in cultures exposed to 3-MB but decreased when exposed to the other three volatiles studied.

The in Vitro Conversion of Norsolorinic Acid to Aflatoxin B1. An Improved Method of Cell-Free Enzyme Preparation and Stabilization

The biosynthesis of the environmental carcinogen aflatoxin B1 (13) is initiated by the formation of a C6-primer by a dedicated yeast-like fatty acid synthase. Homologation of this starter unit by a polyketide synthase gives the anthraquinone norsolorinic acid (2). Approximately 15 chemical steps follow from this first stable intermediate to the mycotoxin (13) itself. A new protocol of cell-free enzyme preparation has been developed from the fungus Aspergillus parasiticus which carries out all of these transformations for the first time. The key experimental step involves rapid concentration and efficient dialysis by membrane filtration to remove primary and secondary metabolites, cofactors, and small biomolecules (MW < 10 000). All enzymes of the aflatoxin biosynthetic pathway have been dramatically stabilized by this procedure, and the effects of added substrates and cofactors can be assayed against virtually no background reactions. The overall pathway from norsolorinic acid (2) to aflatoxin B1 (13) has been investigated, cofactor requirements defined for each step, and a time-course run in which only versicolorin A (9) and sterigmatocystin (11) were observed to accumulate. The post-bisfuran skeletal rearrangement of versicolorin A (9) to demethylsterigmatocystin (10) was studied in O-methylsterigmatocystin (12) in the presence of D2O and/or d7-glucose or stereospecifically labeled NADPD. Unexpectedly high extents of proton exchange were found in the A ring during this transformation, including at a site of formal reduction. A tentative mechanism is discussed to account for this multi enzyme process.

Analysis of mechanisms regulating expression of the ver-1 gene, involved in aflatoxin biosynthesis.

Previous studies have shown that ver-1A encodes an enzyme which is directly involved in the conversion of versicolorin A to demethylsterigmatocystin during aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus. In this study, two different tools were utilized to study the regulation of ver-1A expression at the level of transcription and protein accumulation. First, a ver-1A cDNA was expressed in Escherichia coli with the vector pMAL-c2. The resulting maltose-binding protein-Ver-1A fusion protein was purified and used to generate polyclonal antibodies. Western blot analyses showed that these antibodies specifically recognized the Ver-1 protein (approximately 28 kDa) in cell extracts of Aspergillus parasiticus SU1. Second, a GUS (uidA; encodes beta-glucuronidase) reporter system was developed by fusing the ver-1A promoter and transcription terminator to the GUS gene. Reporter constructs were transformed into A. parasiticus, resulting in a single copy of the ver-1A-GUS reporter integrated adjacent to the wild-type ver-1A gene (3' end) in the chromosome. Western blot analysis, Northern hybridization analysis, and a GUS activity assay were used to analyze transformants. The timing of appearance and pattern of accumulation of GUS transcript and GUS protein in transformants were consistent with the timing of appearance and pattern of accumulation of ver-1 transcript and Ver-1 protein. These data suggested that the GUS gene was under the same regulatory control as the wild-type ver-1 gene and confirmed that transcriptional regulation plays an important role in ver-1A expression. Integration of the ver-1A-GUS reporter construct at the niaD locus resulted in 500-fold-lower GUS activity, but the temporal pattern of accumulation of GUS activity was not affected. Therefore, chromosomal location can play a role in determining the level of gene expression in A. parasiticus and should be an important consideration when analyzing promoter function in this organism.

Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The ver-1A gene was cloned and its nucleotide sequence was determined as part of a previous study on aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus SU-1. A second copy of this gene, ver-1B, was tentatively identified in this fungal strain. In this study, ver-1B was cloned by screening an A. parasiticus cosmid library with a ver-1A probe. The nucleotide sequence of ver-1B was determined. The predicted amino acid sequence of ver-1B had 95% identity with ver-1A. A translational stop codon, found in the ver-1B gene coding region, indicated that it encodes a truncated polypeptide. To confirm the function of the ver-1 genes in AFB1 synthesis, a plasmid (pDV-VA) was designed to disrupt ver-1A and/or ver-1B by transformation of the AFB1 producer A. parasiticus NR-1. One disruptant, VAD-102, which accumulated the pathway intermediate versicolorin A was obtained. Southern hybridization analysis of VAD-102 revealed that ver-1A but not ver-1B was disrupted. A functional ver-1A gene was transformed back into strain VAD-102. Transformants which received ver-1A produced AFB1, confirming that ver-1A is the only functional ver-1 gene in A. parasiticus SU-1 and that its gene product is involved in the conversion of versicolorin A to sterigmatocystin in AFB1 biosynthesis. A duplicated chromosomal region (approximately 12 kb) was identified upstream from ver-1A and ver-1B by Southern hybridization analysis. This duplicated region contained the aflR gene, which is proposed to be one regulator of AFB1, synthesis. A similar gene duplication was also identified in several other strains of A. parasiticus.

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 102 to 2.5 × 104 transformants per 106 viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans.

A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 x 10(2) to 2.5 x 10(4) transformants per 10(6) viable protoplasts and microgram DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.

Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.

A pyruvate decarboxylase gene from Aspergillus parasiticus

A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase.