Elucidation of the functional genomics of antioxidant-based inhibition of aflatoxin biosynthesis

Caffeic acid (3,4-dihydroxycinnamic acid, 12 mM) added to a fat-based growth medium reduces > 95% of aflatoxin production by Aspergillus flavus NRRL 3357, without affecting fungal growth. Microarray analysis of caffeic acid-treated A. flavus indicated expression of almost all genes in the aflatoxin biosynthetic cluster were down-regulated, ranging from a log2 ratio of caffeic acid treated and untreated of − 1.12 (medium) to − 3.13 (high). The only exceptions were genes norB and the aflatoxin pathway regulator-gene, aflJ, which showed low expression levels in both treated and control fungi. The secondary metabolism regulator-gene, laeA, also showed little change in expression levels between the fungal cohorts. Alternatively, expression of genes in metabolic pathways (i.e., amino acid biosynthesis, metabolism of aromatic compounds, etc.) increased (log2 ratio > 1.5). The most notable up-regulation of A. flavus expression occurred in four genes that are orthologs of the Saccharomyces cerevisiae AHP1 family of genes. These genes encode alkyl hydroperoxide reductases that detoxify organic peroxides. These increases ranged from a log2 ratio of 1.08 to 2.65 (moderate to high), according to real-time quantitative reverse transcription-PCR (qRT-PCR) assays. Based on responses of S. cerevisiae gene deletion mutants involved in oxidative stress response, caffeic, chlorogenic, gallic and ascorbic acids were potent antioxidants under oxidative stress induced by organic peroxides, tert-butyl and cumene hydroperoxides. Differential hypersensitivity to these peroxides and hydrogen peroxide occurred among different mutants in addition to their ability to recover with different antioxidants. These findings suggest antioxidants may trigger induction of genes encoding alkyl hydroperoxide reductases in A. flavus. The possibilities that induction of these genes protects the fungus from oxidizing agents (e.g., lipoperoxides, reactive oxygen species, etc.) produced during host-plant infection and this detoxification attenuates upstream signals triggering aflatoxigenesis are discussed.