Activation of Aflatoxin Biosynthesis Alleviates Total ROS in Aspergillus parasiticus

Abstract

An aspect of mycotoxin biosynthesis that remains unclear is its relationship with the cellular management of reactive oxygen species (ROS). Here we conduct a comparative study of the total ROS production in the wild-type strain (SU-1) of the plant pathogen and aflatoxin producer, Aspergillus parasiticus, and its mutant strain, AFS10, in which the aflatoxin biosynthesis pathway is blocked by disruption of its pathway regulator, aflR. We show that SU-1 demonstrates a significantly faster decrease in total ROS than AFS10 between 24 h to 48 h, a time window within which aflatoxin synthesis is activated and reaches peak levels in SU-1. The impact of aflatoxin synthesis in alleviation of ROS correlated well with the transcriptional activation of five superoxide dismutases (SOD), a group of enzymes that protect cells from elevated levels of a class of ROS, the superoxide radicals (O₂^-). Finally, we show that aflatoxin supplementation to AFS10 growth medium results in a significant reduction of total ROS only in 24 h cultures, without resulting in significant changes in SOD gene expression. Our findings show that the activation of aflatoxin biosynthesis in A. parasiticus alleviates ROS generation, which in turn, can be both aflR dependent and aflatoxin dependent.

Keywords: Aspergillus; Key Contribution; This work illustrates how aflatoxin biosynthesis contributes to the management of total ROS in Aspergillus parasiticus; aflR; aflatoxin; aflatoxin biosynthesis; an established model for studying mycotoxin biosynthesis and secondary metabolism in filamentous fungi. We show that activation of aflatoxin biosynthesis reduces total ROS production in this fungus; and aflatoxin itself.; is jointly mediated by the aflatoxin pathway regulator; reactive oxygen species; superoxide dismutase; which at least in part.

Figure 1

Decrease of total ROS during activation of aflatoxin biosynthesis. (a) Comparison of (i) aflatoxin accumulation and (ii) Gene expression levels relative to 24 h of three aflatoxin pathway genes in SU-1 and AFS10. (b) Comparison of total ROS at 24 h and 48 h. The error-bars represent standard error of the mean. The two-tailed p-value was determined using unpaired t-test (GraphPad statistical software). #, Significant difference of transcript levels between 24 h and 48 h (p-value < 0.05, n = 3); * Significant difference of total ROS between SU-1 and AFS10 (p-value < 0.05, n = 3).

Figure 2

Comparison of SOD gene expression in SU-1 and AFS10. Quantitative PCR (qPCR) comparison of SOD gene expression in the two strains at 24 and 48 h of culture growth. All expression quantifications were conducted in triplicate. For each gene the expression value was normalized against the 18s rRNA reference gene and compared to a β-tubulin control. The expression values for each target gene at early stationary phase (48 h) were expressed as the fold change relative to 24 h time point. Fold changes ≥2.0 were considered up- or down-regulated. All data and statistical analysis (Student’s t-test) were performed using CFX Manager software (Bio-Rad Laboratories). Compared to 24 h gene expression, Fesod showed a significant decrease in both the wild-type (2.1-fold; p = 0.003) and AFS10 (3.9-fold; p < 0.001); FesodA showed no significant change for either strain; CuZnsod expression did not change in the WT, but showed a 2.1-fold increase (p = 0.003) in AFS10; CuZnsod1 showed a large, significant decrease in expression for both the WT (22.4-fold; p = 0.001) and AFS10 (26.4-fold; p < 0.001); Mnsod had a dramatically significant 36.2-fold increase in gene expression in the WT (p < 0.001), and an even greater 69.8-fold increase in AFS10 (p < 0.001) compared 24 h expression. (Raw gene expression data is included as Supplementary Figure S1). * Indicates statistically significant difference from respective 24 h gene expression; p ≤ 0.05.

Figure 3

Aflatoxin supplementation to AFS10. (a) Effect on total ROS. A quantitative comparison of ROS in AFS10 supplemented with 50 ppm aflatoxin (in 70% methanol) and a 70% methanol control was conducted. Total ROS was quantified at 24 h and 48 h of growth + 4 h of incubation in 1 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in phosphate buffered saline (PBS) substrate with the corresponding AF concentration. Error-bars represent SEM. (*) denotes statistically significant difference (p < 0.05; n = 3) in ROS compared to the 70% methanol control for the corresponding growth time. (b) Cellular uptake of aflatoxin during aflatoxin supplementation. (i) Percent removal of aflatoxin from the supplementation medium in live cells of 24 h and 48 h AFS10. The percent removal was calculated at every hour until 4 h to compare the aflatoxin removal pattern by live cells with the dead cells that allow free diffusion from the medium into the cells. (ii) Percent aflatoxin accumulation in the mycelium of 24 h and 48 h cultures. Aflatoxin in the mycelia of live cells was compared to the dead cells. Error-bars represent SEM. a, statistically significant difference (p < 0.05; n = 3) in aflatoxin levels with 0 h, b, statistically significant difference (p < 0.05; n = 3) in aflatoxin levels between 24 h and 48 h cultures, c, statistically significant difference (p < 0.05; n = 3) in aflatoxin levels between live and dead cells at a particular time-point. (c) Comparison of SOD gene expression in aflatoxin supplemented and control AFS10. qPCR comparison of SOD gene expression in the control and 4 h aflatoxin supplemented cells. The gene expression values were normalized against the 18s rRNA reference gene. Fold changes ≥2.0 were considered up- or down-regulated. All data and statistical analysis (Student’s t-test) were performed using CFX Manager software (Bio-Rad Laboratories).

Figure 4

Proposed model for total ROS management in A. parasiticus. Based on our current findings and previous reports we propose that aflatoxin-dependent protection occurs in one or a combination of the following ways: (a) utilization of ROS in the biochemical steps of the biosynthesis pathway [33], (b) aflatoxin-dependent reduction of ROS in cells at exponential growth phase (current study) and (c) aflR-dependent reduction of ROS (current study) possibly through its gene regulatory impacts outside the aflatoxin pathway gene cluster [43,44]. Aflatoxin-dependent biochemical processes that sequester ROS still remain uncharacterized (green dashed arrow). Pink arrows indicate the sources of ROS accumulation. These include ROS generation from primary metabolic processes, secondary ROS generated from aflatoxin biosynthesis [45], and ROS generated upon aflatoxin uptake by cells during stationary phase of growth (based on aflatoxin supplementation data from 48 h AFS10 cultures in the current study). The mechanisms that result in ROS accumulation upon cellular uptake of aflatoxin remains uncharacterized (pink dashed arrow). The model can now explain the physiological need of the cells to co-regulate secondary metabolism (in this case, aflatoxin biosynthesis) and oxidative stress response through the bZIP proteins [3,24,25,26,27,28,29,30,31]. Red arrows indicate the contributions of the current study. The molecular mechanism of aflR-mediated regulation of SOD genes remains uncharacterized (red dashed arrow) and will be investigated in our follow up studies.