Modulation of antioxidant defense in Aspergillus parasiticus is involved in aflatoxin biosynthesis: a role for the ApyapA gene

Abstract

Oxidative stress is recognized as a trigger of different metabolic events in all organisms. Various factors correlated with oxidation, such as the beta-oxidation of fatty acids and their enzymatic or nonenzymatic by-products (e.g., precocious sexual inducer factors and lipoperoxides) have been shown to be involved in aflatoxin formation. In the present study, we found that increased levels of reactive oxygen species (ROS) were correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus. To better understand the role of ROS formation in toxin production, we generated a mutant (Delta ApyapA) having the ApyapA gene deleted, given that ApyapA orthologs have been shown to be part of the antioxidant response in other fungi. Compared to the wild type, the mutant showed an increased susceptibility to extracellular oxidants, as well as precocious ROS formation and aflatoxin biosynthesis. Genetic complementation of the Delta ApyapA mutant restored the timing and quantity of toxin biosynthesis to the levels found in the wild type. The presence of putative AP1 (ApYapA orthologue) binding sites in the promoter region of the regulatory gene aflR further supports the finding that ApYapA plays a role in the regulation of aflatoxin biosynthesis. Overall, our results show that the lack of ApyapA leads to an increase in oxidative stress, premature conidiogenesis, and aflatoxin biosynthesis.

FIG. 1.

DNA gel blot analysis of ApyapA gene replacement mutants and complementation. (A) WT ApyapA locus (ApY); final deletion event with construct containing acetamide resistance cassette (AmdS) and ApyapA gene XhoI-EcoRI (X-E) and SphI-SalI (Sp-Sa) fragments used for transforming WT protoplast; the 5.4-kb BglII-HindIII (B-H) fragment which carries also the hygromycin B resistance cassette (hph) used for complementing ΔApyapA strains. The probe used for the subsequent Southern blot analysis is indicated (p). Genomic DNA was isolated from the wild-type strain NRRL 2999 (WT), the ApyapA complemented mutant (CM), and the gene replacement transformant (M) and digested with EcoRI (E). (B) The blots were hybridized with 1.9-kb ApyapA PCR DIG-labeled probes. (C) PCR amplification of WT, CM, and M strain genomic DNA using AmdS_for and ApyapA_rev primers (expected size of the PCR fragment, ∼2.1 kb) or AmdS_for and AmdS_rev primers (expected size of the PCR fragment, ∼0.7 kb) or Hph_for and Hph_rev primers (expected size of the PCR fragment, ∼0.65 kb) or Hph_for and ApyapA_rev2 primers (expected size of the PCR fragment, ∼0.9 kb). The numbers in the “keys” column indicate the primers used for PCR amplification.

FIG. 2.

(A) Mycelial growth (mg [dry weight]·ml⁻¹) of WT, ΔApyapA (M), and ApyapA complemented (CM) strains inoculated in PDB (25 ml) and incubated at 30°C from 12 up to 168 h. (B) Numbers of conidia produced by WT, M, and CM strains cultured under the same experimental conditions. (C) Agar plates (PDA) showing the different timing in conidium formation of WT, M, and CM strains at different time intervals after inoculation (24 to 168 h). The results in panels A and B are the means ± SEMs of three determinations from three separate experiments.

FIG. 3.

(A) Detection of superoxide anion formation (reported as absorbance of XTT formazan at 490 nm). (B) H₂O₂ formation (μmol) in mycelia of WT and ΔApyapA (M) strains grown in PDB (25 ml) and incubated at 30°C from 8 to 168 h. The results are the means ± SEMs of three determinations from three separate experiments.

FIG. 4.

(A) 9- and 13-HODE (ng·mg⁻¹ [dry weight]). (B) LOX-like activity measured as diene conjugate formation at 234 nm (U·mg⁻¹ protein) in mycelia of WT and ΔApyapA (M) strains grown in PDB (25 ml) and incubated at 30°C from 10 to 168 h. The results are the means ± SEMs of three determinations from three separate experiments.

FIG. 5.

RT-PCR analysis of oxidative stress transcription factor (ApyapA, Apskn7, and Aphsf2) mRNA in A. parasiticus mycelia from WT and M strains, grown in PDB after different periods of incubation. The results are representative of three separate experiments.

FIG. 6.

Antioxidant enzyme SOD, GPX (U·mg⁻¹ protein), and HPR (absorbance at 234 nm min⁻¹·mg⁻¹ protein) activities in mycelia of A. parasiticus WT and ΔApyapA (M) strains grown in PDB at different time intervals of incubation at 30°C. (A and B) SOD activity at pH 7.8 (A) and pH 10.0 (B); (C) GPX activity; (D1) HPR activity; (D2) zymogram analysis of WT and M strain protein extracts obtained from mycelia grown for different time periods at 30°C in PDB and fractionated in a native polyacrylamide gel stained for CAT activity. C1 represents a low-acidic conformer of CAT derived from A. niger, and C2 represents the same CAT oxidized under an O₂ stream, giving a more-acidic form. The dashed white line represents the trend (from low- to high-acidic form) of CAT conformers during the different time intervals. The results in panels A to D1 are the means ± SEMs of three determinations from three separate experiments.

FIG. 7.

(A to D) Ratios between the fungal growth (mg [dry weight]·ml⁻¹) of WT and ΔApyapA (M) strains in CD amended with CH (1 mM) (A) or 1 (B) or 10 (C) mM H₂O₂ or Men (0.5 mM) (D) and their growth in CD (control). (E) Aflatoxin (AFTX) production in culture filtrate (μg·ml⁻¹) of WT and M strains grown in media amended with different stressors (CH, 1 mM; Men, 0.5 mM; H₂O₂, 1 and 10 mM), at different time intervals (4 to 11 days). All the compounds were added after 4 days of incubation at 30°C in CD medium. The results are the means ± SEMs of three determinations from three separate experiments.

FIG. 8.

(A) Aflatoxin (AFTX) production in culture filtrate (μg ml⁻¹) of A. parasiticus WT, ΔApyapA (M), and complemented ApyapA (CM) strains inoculated into aflatoxin-conducive medium (PDB). (B) aflR and norA mRNA RT-PCR analysis in A. parasiticus mycelia after different time intervals. The aflatoxin results are the means ± SEMs of three determinations from three separate experiments.