Volatile profiling reveals intracellular metabolic changes in Aspergillus parasticus: veA regulates branched chain amino acid and ethanol metabolism

Background: Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results: Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions: 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and b-oxidation of fatty acids. 3) Intracellular chemical development in A. parasiticus is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products

Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

<p>Abstract</p> <p>Background</p> <p>Filamentous fungi in the genus <it>Aspergillus </it>produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by <it>Aspergillus parasiticus </it>in an attempt to define the association of secondary metabolism with other metabolic and cellular processes.</p> <p>Results</p> <p>Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain <it>Aspergillus parasiticus </it>SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator <it>veA </it>affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a <it>veA </it>disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation.</p> <p>Conclusions</p> <p>1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and β-oxidation of fatty acids. 3) Intracellular chemical development in <it>A. parasiticus </it>is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products.</p

Role of cis-Acting Sites NorL, a TATA Box, and AflR1 in nor-1 Transcriptional Activation in Aspergillus parasiticus

The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus Aspergillus. nor-1 encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (AflR1). Proteins in Aspergillus parasiticus cell extracts and AflR expressed in Escherichia coli do not bind to A. parasiticus AflR1 in vitro, so it was not clear if this site was required for nor-1 expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in nor-1 expression in A. parasiticus and identified additional cis-acting sites required for maximum nor-1 transcriptional activation. Deletion and substitution of AflR1 in the nor-1 promoter in A. parasiticus nor-1::GUS reporter strains showed that this site is required for nor-1 transcriptional activation in vivo. Substitution of a putative TATA box in the nor-1 promoter resulted in nondetectable β-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel cis-acting site, designated NorL, between residues −210 and −238 that was required for maximum nor-1 transcriptional activation in A. parasiticus grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific NorL-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the NorL site appears only once in the aflatoxin gene cluster, its association with the nor-1 promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway

The Fungal bZIP Transcription Factor AtfB Controls Virulence-Associated Processes in Aspergillus parasiticus

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