Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro. Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis. We generated A. parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli. Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA. Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo. Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development. We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation. OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract. OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed. OmtA in older fractions of the colony (24 to 72 h) was partly degraded. Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores. These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age.     

Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of<Emphasis Type="Italic"> Aspergillus parasiticus</Emphasis>

The biosynthesis of aflatoxin in Aspergillus parasiticus is a complex process that involves the activities of at least 18 pathway enzymes. The distribution of these enzymes within fungal colonies and fungal cells is not clearly understood. The objective of this study was to investigate the distribution and subcellular location of Nor-1, Ver-1, and OmtA, which represent early, middle, and late enzymatic activities, respectively, in the aflatoxin biosynthetic pathway. The distribution of these three enzymes within A. parasiticus SU-1 was analyzed in time-fractionated, 72-h fungal colonies (fraction 1, 48–72 h; fraction 2, 24–48 h; fraction 3, 0–24 h). Western blot analysis and immunofluorescence microscopy demonstrated the highest abundance of Nor-1, Ver-1, and OmtA in colony fraction 2. Fungal tissues in this fraction were analyzed by immunoelectron microscopy. Nor-1 and Ver-1 were primarily localized to the cytoplasm, suggesting that they are cytosolic enzymes. OmtA was also detected in the cytoplasm. However, in cells located near the basal (substrate) surface of the colony, OmtA was predominantly detected in organelles tentatively identified as vacuoles. The role of this organelle in toxin biosynthesis is unclear. The relative distribution of OmtA to the cytoplasm or to vacuole-like organelles may depend on the age and/or physiological condition of the fungal cells.     

veA is required for toxin and sclerotial production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway.

The penultimate step in the aflatoxin biosynthetic pathway of the filamentous fungi Aspergillus flavus and A. parasiticus involves conversion of sterigmatocystin to O-methylsterigmatocystin. An S-adenosylmethionine-dependent methyltransferase that catalyzes this reaction was purified to homogeneity (> 90%) from 78-h-old mycelia of A. parasiticus SRRC 163. Purification of this soluble enzyme was carried out by five soft-gel chromatographic steps: cell debris remover treatment, QMA ACELL chromatography, hydroxylapatite-Ultrogel chromatography, DEAE-Spherodex chromatography, and Octyl Avidgel chromatography, followed by MA7Q high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein peak from this step on silver staining identified a single band of approximately 40 kDa. This purified protein was distinct from the dimeric 168-kDa methyltransferase purified from the same fungal strain under identical growth conditions (D. Bhatnagar, A. H. J. Ullah, and T. E. Cleveland, Prep. Biochem. 18:321-349, 1988). The chromatographic behavior and N-terminal sequence of the 40-kDa enzyme were also distinct from those of the 168-kDa methyltransferase. The molar extinction coefficient of the 40-kDa enzyme at 278 nm was estimated to be 4.7 x 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5).     

Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.     

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures.

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.     

Evidence for de novo synthesis of an aflatoxin pathway methyltransferase near the cessation of active growth and the onset of aflatoxin biosynthesis in Aspergillus parasiticus mycelia

The accumulation of both activity and protein of a methyltransferase (MTase) from Aspergillus parasiticus, which catalyzes conversion of sterigmatocystin to O-methylsterigmatocystin in the aflatoxin pathway, was detected in fungal mycelia slightly before the onset of aflatoxin biosynthesis in the same cultures. MTase protein was identified in mycelial postmicrosomal (soluble protein) fractions by electrophoresis and subsequent immunoblotting using antiserum raised against purified MTase protein; MTase activity was determined by measuring the rate of conversion of sterigmatocystin to O-methylsterigmatocystin in the presence of soluble protein fractions. Using the above technique, it was determined that MTase protein as well as MTase activity increased sharply in mycelia 30 to 45 h after inoculation, shortly after which, mycelial growth rate began to decline. During the subsequent time interval (45 to 70 h after inoculation), a sharp increase in aflatoxin levels was detected in the culture medium. Results obtained from an experiment in which cycloheximide was added to cultures at various times to inhibit protein synthesis and from an experiment in which mycelial proteins were radiolabelled to identify newly synthesized proteins indicated that accumulation of MTase activity and protein in late growth phase mycelia is due to de novo protein synthesis.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

veA Is Required for Toxin and Sclerotial Production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

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.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species.

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Involvement of two cytosolic enzymes and a novel intermediate, 5'-oxoaverantin, in the pathway from 5'-hydroxyaverantin to averufin in aflatoxin biosynthesis

During aflatoxin biosynthesis, 5'-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5'-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A. parasiticus by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A. parasiticus. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed.     

Fate of the methyl group during the conversion of sterigmatocystin into O-methylsterigmatocystin and aflatoxin B1 by cell-free preparations of Aspergillus parasiticus

Cell-free extracts of fungal mycelia of two aflatoxin non-producing isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were utilized for the study of enzyme activities involved in the latter stages of aflatoxin biosynthesis. The post-microsomal fractions (105,000 x g supernatant) of both SRRC 163 and SRRC 2043 were able to convert sterigmatocystin (ST) into O-methylsterigmatocystin (OMST); whereas the microsomal (105,000 x g pellet) preparation of only SRRC 163 was able to convert OMST into aflatoxin B1 (AFB1). S-Adenosylmethionine (SAM) was the primary substrate for the ST to OMST (methyltransferase) enzymatic conversion; [3H]OMST of specific activity 0.93 Ci/mmol was obtained in a reaction containing the [3H]SAM substrate (specific activity 1 Ci/mmol). After the terminal enzymatic conversion of OMST into AFB1, none of the radiolabel of the methyl group from OMST was found in AFB1. It is postulated that the methylation of ST may be required for subsequent enzymatic oxidation of OMST to aflatoxin B1.