Evidence that a transcription factor regulatory network coordinates oxidative stress response and secondary metabolism in aspergilli

Abstract

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.

Figure 1

Promoter regions of aflatoxin biosynthetic and antioxidant genes used in electrophoretic mobility shift assays. (A) Schematic representation of the relevant genes in the aflatoxin gene cluster. (B) Schematic of fas-2/fas-1 intergenic region. The fas-2/fas-1 intergenic region was divided into two fragments designated Fas2 and Fas1. The Fas1 fragment was further subdivided into two smaller fragments designated Fas1-b and Fas1-a. Five putative cis-acting sites are shown including CRE and stress-response element (STRE) sites. The position of a 28-bp Fas competitor is shown. (C) Schematic of norA/ver-1 intergenic region. The norA/ver-1 intergenic region was divided into two fragments designated Ver1-b and Ver1-a. Six putative cis-acting sites are shown including CRE and STRE sites. The position of 55-bp Ver1 and Ver1 m competitors is shown. (D) Schematic of omtA/ordA intergenic region. The omtA/ordA intergenic region was divided into two fragments designated OmtA and OrdA. Ten putative cis-acting sites are shown including CRE and STRE sites. (E) Schematic of mycelial cat1 promoter region designated Mcat1. The mycelial cat1 promoter region was divided into two fragments designated Mcat1-b and Mcat1-a. Five putative cis-acting sites are shown including CRE and STRE sites. (F) Schematic of Mn sod promoter region designated Msod. Five putative cis-acting sites are shown including CRE and STRE sites.

Figure 2

Time-course of DNA–protein complex formation as detected by electrophoretic mobility shift assays using aflatoxin (fas-1 and ver-1) and antioxidant gene (mycelial cat1 and Mn sod) promoters. Aspergillus parasiticus SU-1 was grown for 24, 48, or 60 h at 30°C in YES medium. Cell extracts enriched in nuclear proteins were prepared as described in Experimental Procedures. Five micrograms of enriched nuclear protein extracts was added to a P³²-labeled promoter probes for each aflatoxin or antioxidant gene. (A) Fas1-a, Ver1-b, and OmtA probes. (B) Mcat1-a and Msod probes.

Figure 3

Competition electrophoretic mobility shift assays. Aspergillus parasiticus SU-1 was grown for 48 h at 30°C in YES medium. (A) Fas1-a and Ver1-b probes. (B) Mcat1-a and Msod probes. The 51-bp nonlabeled NorR4 fragment (contains CRE and AP-1 sites; 50- and 250-fold molar excess) was added to compete for labeled Fas1-a, Ver1-b, Mcat1-a, or Msod probes. (C) Fas1-b, Fas1-a, and Ver1-b probes. The 28-bp nonlabeled Fas fragment (contains 8-base motif, 5′-AGCCG/CTG/CA/G-3′; 50- and 250-fold molar excess) was added to compete for labeled Fas1-b, Fas1-a, or Ver1-b probes.

Figure 4

Electrophoretic mobility shift assays of AtfB binding in the fas-1, ver-1, omtA, vbs, mycelial cat1, and Mn sod promoters. Aspergillus parasiticus SU-1 was grown for 24, 48, or 60 h at 30°C in YES medium. Enriched nuclear protein extracts were prepared as described in Experimental Procedures. Five micrograms of enriched nuclear protein extracts was added to a P³²-labeled promoter probe for each aflatoxin or antioxidant gene. Anti-AtfB antibodies (YSR) or preimmune serum was added to determine whether these could block protein/DNA interaction (shift inhibition). (A) Fas1-a probe. Nuclear protein extracts were used from 24-, 48-, or 60-h culture. (B) Ver1-b probe. Nuclear protein extracts were used from 24-, 48-, or 60-h culture. (C) OmtA probe. Nuclear protein extracts were used from 48-h culture. (D) Vbs probe. Nuclear protein extracts were used from 48-h culture. (E) Mcat1-a probe. Nuclear protein extracts were used from 24-, 48-, or 60-h culture. (F) Msod probe. Nuclear protein extracts were used from 24-, 48-, or 60-h culture.

Figure 5

Sequence-based motif analysis of fas-1, ver-1, mycelial cat1, and Mn sod promoters using MEME. Fas1-a, Ver1-b, Mcat1-a, and Msod promoters fragment and the NorR4 competitor used for competition electrophoretic mobility shift assays were analyzed for conserved motifs using sequence-based MEME motif analysis. A conserved motif (5′-T/GNT/CAAGCCNNG/AA/GC/ANT/C-3′) was found in the promoter regions, where the core consensus sequence was 5′-AAGCC-3′. (A) Sites carrying the core consensus sequence in five promoter fragments. (B) Sequence logo of the core consensus sequence. (C) Sites carrying the conserved motif in five promoter fragments. (D) Sequence logo of the conserved motif.

Figure 6

Competition electrophoretic mobility shift assays of fas-1, ver-1, mycelial cat1, and Mn sod promoters using a 55-bp Ver1 promoter fragment as a competitor. Aspergillus parasiticus SU-1 was grown for 24 or 60 h at 30°C in YES medium. The 55-bp nonlabeled Ver1 promoter fragment (contains both stress-response element and CRE sites; 50- and 250-fold molar excess) was added to compete for labeled Fas1-a, Ver1-b, Mcat1-a, or Msod probes. (A) Fas1-a probe. (B) Ver1-b probe. (C) Mcat1-a probe. (D) Msod probe.

Figure 7

Time-course of DNA–protein complex formation as detected by electrophoretic mobility shift assays using oligonucleotide probes carrying a stress-response element (STRE) site. Aspergillus parasiticus SU-1 was grown for 24, 48, or 60 h at 30°C in YES medium. Enriched nuclear protein extracts were prepared as described in Experimental Procedures. Five micrograms of enriched nuclear protein extracts was added to the P³²-labeled 55-bp Ver1 (contains STRE and CRE sites) or Ver1 m (contains STRE site) oligonucleotide probes in each lane.

Figure 8

Expression of fas-1, ver-1, mycelial cat1, Mn sod, atfB, AP-1, and MsnA. Aspergillus parasiticus SU-1 was grown for 24, 48, or 60 h at 30°C in YES medium. Total RNA extraction and real-time PCR analyses were performed as described in Experimental Procedures. The relative level of mRNA is represented as the mRNA level of the target gene divided by the mRNA level of β-tubulin at the same time point. Bars represents mean ± SE (n = 4). Statistical analysis was performed by one-way repeated measure analysis of variance. Same lowercase letter indicates that there is no statistically significant difference between two measurements. Different lowercase letters specify a statistically significant difference between two measurements. P values are as follows: fas-1 and ver-1 (P = 0.002), mycelial cat1 and atfB (P = 0.011), Mn sod (P < 0.001), and AP-1 and MsnA (P = 0.015). Two independent biological replicates were performed showing the same trend. Two duplicates samples were analyzed for each biological replicate.

Figure 9

Proposed model for transcriptional activation of secondary metabolism and oxidative stress-response genes by binding of transcription factors in response to oxidative stress. (A) Schematic representation of the transcription factor-binding sites in the promoters of the aflatoxin biosynthetic and antioxidant genes used in this study. (B) Regulatory network for transcriptional activation of secondary metabolism and oxidative stress-response genes for cellular defense against oxidative stress. Based on available experimental evidence, we propose that increased levels of intracellular ROS in fungal cells downregulate the cAMP-PKA signaling pathway. This promotes MsnA binding to stress-response element (STRE) sites in promoters of oxidative stress-response (OR) genes including antioxidant genes for their activation. Simultaneously, ROS upregulates the SAPK signaling cascades. This promotes AtfB and SrrA binding (SrrA recruits AP-1) to corresponding CRE, SRRA, and AP1 sites in promoters of the oxidative stress-response (OR) genes including the antioxidant genes for their induction. Then, MsnA, AtfB, and SrrA bind (SrrA recruits AP-1) to corresponding STRE, CRE, SRRA, and AP1 sites in promoters of secondary metabolism genes including aflatoxin genes for their activation due to excess ROS. Secondary metabolites produced in response to ROS play a role in a defense mechanism of fungal cells against oxidative stress. PKA, protein kinase A; SAPK, stress-activated protein kinase; OR, oxidative stress response; SM, secondary metabolism.