Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time

Abstract

Background: The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time.

Results: Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15 mM H₂O₂, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which encompassed 9 biological super-pathways and 47 sub-pathways. These metabolites were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points.

Conclusions: The moderately high aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H₂O₂-stressed A. flavus and drought-stressed hosts also suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.

Keywords: Aflatoxin; Aspergillus flavus; Drought stress; Metabolomics; Oxidative stress.

Fig. 1

Growth curve analysis of Aspergillus flavus isolates AF13 and NRRL3357 under increasing oxidative stress and conidial concentration. The growth of AF13 (a, c) and NRRL3357 (b, d) were examined under increasing H₂O₂ concentrations in YES medium inoculated with either 2.0 × 10⁴ (a, b) or 8.0 × 10⁴ conidia/mL (c, d) by monitoring absorbance at 405 nm over 100 h. A threshold of 0.2 was selected for growth initiation timing which corresponded with linear phase initiation for both isolates in most conditions and is indicated by the dashed red line. Error bars represent standard deviation. No growth was detected at H₂O₂ concentrations > 15 mM in NRRL3357 with earlier growth initiation detected at higher conidia concentrations

Fig. 2

Principal components analysis (PCA) of metabolite accumulation. A4N, A4Y, A7N, and A7Y refer to AF13 at 4 and 7 DAI with and without 15 mM H₂O₂ treatment. N4N, N4Y, N7N, and N7Y refer to the same for NRRL3357. Dark blue points correspond with AF13 with no stress and light blue points refer to AF13 with stress. Orange points correspond with NRRL3357 with no stress and light orange points refer to NRRL3357 with stress. Circles represent samples at 4 DAI and triangles represent samples at 7 DAI

Fig. 3

Differential accumulation of compounds involved in carbohydrate metabolism, glutathione metabolism, and amino acid biosynthesis. Heatmaps located at each metabolite represent the changes in metabolite accumulation in response to oxidative stress in AF13 and NRRL3357 at 4 and 7 DAI. Red and green indicate significant increases and decreases in metabolite levels, respectively (p < 0.05). Light red and light green indicate marginally significant increases and decreases in metabolite levels, respectively (0.05 < p < 0.10). Grey represents no significant changes

Fig. 4

Differential accumulation of compounds involved in polyamine and sulfur metabolism. Heatmaps located at each metabolite represent the changes in metabolite accumulation in response to oxidative stress in AF13 and NRRL3357 at 4 and 7 DAI. Red and green indicate significant increases and decreases in metabolite levels, respectively (p < 0.05). Light red and light green indicate marginally significant increases and decreases in metabolite levels, respectively (0.05 < p < 0.10). Grey represents no significant changes

Fig. 5

Differential accumulation of compounds involved in lipid metabolism. Heatmaps located at each metabolite represent the changes in metabolite accumulation in response to oxidative stress in AF13 and NRRL3357 at 4 and 7 DAI. Red and green indicate significant increases and decreases in metabolite levels, respectively (p < 0.05). Light red and light green indicate marginally significant increases and decreases in metabolite levels, respectively (0.05 < p < 0.10). Grey represents no significant changes