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Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B 1 (AFB 1 ), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB 1 removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB 1 removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB 1 analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB 1 removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24-48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB 1 removal activity by whole cells was mainly due to AFB 1 binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB 1 binding process onto cell wall components.

Fungal basic leucine zipper (bZIP) transcription factors mediate responses to oxidative stress. The ability to regulate stress response pathways in Aspergillus spp. was postulated to be an important virulence-associated cellular process, because it helps establish infection in humans, plants, and animals. Previous studies have demonstrated that the fungal transcription factor AtfB encodes a protein that is associated with resistance to oxidative stress in asexual conidiospores, and AtfB binds to the promoters of several stress response genes. Here, we conducted a gene silencing of AtfB in Aspergillus parasiticus , a well-characterized fungal pathogen of plants, animals, and humans that produces the secondary metabolite and carcinogen aflatoxin, in order to determine the mechanisms by which AtfB contributes to virulence. We show that AtfB silencing results in a decrease in aflatoxin enzyme levels, the down-regulation of aflatoxin accumulation, and impaired conidiospore development in AtfB-silenced strains. This observation is supported by a decrease of AtfB protein levels, and the down-regulation of many genes in the aflatoxin cluster, as well as genes involved in secondary metabolism and conidiospore development. Global expression analysis (RNA Seq) demonstrated that AtfB functionally links oxidative stress response pathways to a broader and novel subset of target genes involved in cellular defense, as well as in actin and cytoskeleton arrangement/transport. Thus, AtfB regulates the genes involved in development, stress response, and secondary metabolism in A. parasiticus . We propose that the bZIP regulatory circuit controlled by AtfB provides a large number of excellent cellular targets to reduce fungal virulence. More importantly, understanding key players that are crucial to initiate the cellular response to oxidative stress will enable better control over its detrimental impacts on humans., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Ochratoxin A (OTA), a mycotoxin, contaminates agricultural products and poses a serious threat to public health worldwide. Microbiological methods are known to be a promising approach for OTA biodegradation because physical and chemical methods have practical limitations. In the present study, a total of 130 fungal isolates obtained from 65 traditional Korean meju (a fermented starter for fermentation of soybeans) samples were examined for OTA-biodegradation activity using thin-layer chromatography. Two fungal isolates were selected for OTA-biodegradation activity and were identified as Aspergillus tubingensis M036 and M074 through sequence analysis of the beta-tubulin gene. After culturing both A. tubingensis isolates in Soytone-Czapek medium containing OTA (40 ng/ml), OTA-biodegradation activity was analyzed using high-performance liquid chromatography (HPLC). Both A. tubingensis strains degraded OTA by more than 95.0% after 14 days, and the HPLC analysis showed that the OTA biodegradation by the A. tubingensis strains led to the production of ochratoxin α, which is much less toxic than OTA. Moreover, crude enzymes from the cultures of A. tubingensis M036 and M074 led to OTA biodegradation of 97.5% and 91.3% at pH 5, and 80.3% and 75.3% at pH 7, respectively, in a buffer solution containing OTA (40 ng/ml) after 24 h. In addition, the OTA-biodegrading fungi did not exhibit OTA production activity. Our data suggest that A. tubingensis isolates and their enzymes have the potential for practical application to reduce levels of OTA in food and feed.

Aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus involves a minimum of 21 enzymes, encoded by genes located in a 70 kb gene cluster. For aflatoxin biosynthesis to be completed, the required enzymes must be transported to specialized early and late endosomes called aflatoxisomes. Of particular significance, seven aflatoxin biosynthetic enzymes are P450/monooxygenases which catalyze reactions that can produce reactive oxygen species (ROS) as byproducts. Thus, oxidative reactions in the aflatoxin biosynthetic pathway could potentially be an additional source of intracellular ROS. The present work explores the hypothesis that the aflatoxin biosynthetic pathway generates ROS (designated as "secondary" ROS) in endosomes and that secondary ROS possess a signaling function. We used specific dyes that stain ROS in live cells and demonstrated that intracellular ROS levels correlate with the levels of aflatoxin synthesized. Moreover, feeding protoplasts with precursors of aflatoxin resulted in the increase in ROS generation. These data support the hypothesis. Our findings also suggest that secondary ROS may fulfill, at least in part, an important mechanistic role in increased tolerance to oxidative stress in germinating spores (seven-hour germlings) and in regulation of fungal development.

The mycotoxin aflatoxin is a secondary metabolite and potent human carcinogen. We investigated one mechanism that links stress response with coordinate activation of genes involved in aflatoxin biosynthesis in Aspergillus parasiticus. Electrophoretic mobility shift assays demonstrated that AtfB, a basic leucine zipper (bZIP) transcription factor, is a master co-regulator that binds promoters of early (fas-1), middle (ver-1), and late (omtA) aflatoxin biosynthetic genes as well as stress-response genes (mycelia-specific cat1 and mitochondria-specific Mn sod) at cAMP response element motifs. A novel conserved motif 5'-T/GNT/CAAG CCNNG/AA/GC/ANT/C-3' was identified in promoters of the aflatoxin biosynthetic and stress-response genes. A search for transcription factors identified SrrA as a transcription factor that could bind to the motif. Moreover, we also identified a STRE motif (5'-CCCCT-3') in promoters of aflatoxin biosynthetic and stress-response genes, and competition EMSA suggested that MsnA binds to this motif. Our study for the first time provides strong evidence to suggest that at least four transcription factors (AtfB, SrrA, AP-1, and MsnA) participate in a regulatory network that induces aflatoxin biosynthesis as part of the cellular response to oxidative stress in A. parasiticus., (© 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Aflatoxin is among the most potent naturally occurring carcinogens known. Previous studies demonstrated that endosomes in the filamentous fungus Aspergillus parasiticus carry enzymes that catalyze the final two steps in aflatoxin synthesis, and these structures also play a role in aflatoxin storage and export. We hypothesized that endosomes house a complete and functional aflatoxin biosynthetic pathway. To address this hypothesis, we purified a cellular fraction containing endosomes, transport vesicles, and vacuoles (V fraction) from A. parasiticus grown under aflatoxin inducing and noninducing conditions. We also added (fed) aflatoxin pathway intermediates to V fraction to test the functional status of aflatoxin pathway enzymes. High throughput LC-MS/MS analysis of proteins in V fraction detected 8 aflatoxin enzymes with high reliability and 8 additional enzymes at lower reliability, suggesting that most aflatoxin pathway enzymes are present. Purified V fraction synthesized aflatoxin and addition of the pathway intermediate versicolorin A increased aflatoxin synthesis, confirming that middle and late aflatoxin enzymes in V fraction are functional. Of particular significance, proteomic and biochemical analysis strongly suggested that additional secondary metabolic pathways as well as proteins involved in response to heat, osmotic, and oxidative stress are housed in V fraction.

Aflatoxin biosynthesis in Aspergillus parasiticus requires at least 17 enzyme activities (from acetate). Although the activities of most aflatoxin biosynthetic enzymes have been established, the mechanisms that govern transport and sub-cellular localization of these enzymes are not clear. We developed plasmid constructs that express Nor-1 fused to a green fluorescent protein reporter (EGFP) to monitor transport and localization of this early pathway enzyme in real time in Aspergillus parasiticus. Plasmids expressing EGFP fused to Nor-1 were introduced into A. parasiticus B62 (carries non-functional Nor-1). Transformants were screened for increased aflatoxin accumulation (restored Nor-1 activity) on coconut agar medium and for EGFP expression using fluorescence microscopy. Increased aflatoxin accumulation was confirmed by TLC and ELISA. Nor-1 fused to EGFP at either the N- or C- terminus functionally complemented non-functional Nor-1 in B62 and increased aflatoxin synthesis to wild-type (N-terminus) or lower levels (C-terminus). We detected full-length Nor-1 fusion proteins in transformants with increased aflatoxin accumulation (Western blot) and determined that the expression plasmid integrated at the nor-1 locus in these cells (Southern blot). Confocal laser scanning microscopy (CLSM) demonstrated that Nor-1 fusion proteins localized in the cytoplasm and vacuoles of fungal hyphae grown on aflatoxin-inducing solid media for 48h; control EGFP (no Nor-1) did not localize to vacuoles until 72h. The highest rate of aflatoxin synthesis coincided with the highest rate of transport of Nor-1 fusion proteins to the vacuole strongly suggesting that Nor-1 is synthesized in the cytoplasm and transported to the vacuole to carry out an early step in aflatoxin synthesis.

Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.

The 27 genes involved in aflatoxin biosynthesis are clustered within a 70 kb region in the Aspergillus parasiticus genome. Using chromatin immunoprecipitation, we demonstrated a positive correlation between the initiation and spread of histone H4 acetylation in aflatoxin promoters and the onset of accumulation of aflatoxin proteins and aflatoxin. Histone H4 acetylation in the pksA (encodes an 'early' biosynthetic pathway enzyme) promoter peaked at 30 h, prior to the increased acetylation in the omtA and ordA (encode 'late' enzymes) promoters detected at 40 h. The specific order in which pksA, ver-1 (encodes a 'middle' enzyme) and omtA transcripts accumulated in cells paralleled the pattern of spread of histone H4 acetylation. Binding of AflR, a positive regulator of aflatoxin biosynthesis, to the ordA promoter showed a positive correlation with the spread of histone H4 acetylation. The data suggest that the order of genes within the aflatoxin cluster determines the timing and order of transcriptional activation, and that the site of initiation and spread of histone H4 acetylation mediate this process. Our data indicate that the aflatoxin and adjacent sugar utilization clusters are part of a larger 'regulatory unit'.