Menadione-Induced Oxidative Stress Re-Shapes the Oxylipin Profile of Aspergillus flavus and Its Lifestyle

Abstract

Aspergillus flavus is an efficient producer of mycotoxins, particularly aflatoxin B₁, probably the most hepatocarcinogenic naturally-occurring compound. Although the inducing agents of toxin synthesis are not unanimously identified, there is evidence that oxidative stress is one of the main actors in play. In our study, we use menadione, a quinone extensively implemented in studies on ROS response in animal cells, for causing stress to A. flavus. For uncovering the molecular determinants that drive A. flavus in challenging oxidative stress conditions, we have evaluated a wide spectrum of several different parameters, ranging from metabolic (ROS and oxylipin profile) to transcriptional analysis (RNA-seq). There emerges a scenario in which A. flavus activates several metabolic processes under oxidative stress conditions for limiting the ROS-associated detrimental effects, as well as for triggering adaptive and escape strategies.

Keywords: LC-MS/MS; RNA-seq; menadione; oxidative stress; oxylipins.

Figure 1

Fold induction/decrease of menadione (Men)-treated vs. untreated samples pinpointed in a color scale. The fungal growth (mg/mL), conidiogenesis (number of conidia/mL), aflatoxin B₁ production (ppb), catalases (CAT and superoxide dismutases (SOD) activities (U/mg proteins), hydrogen peroxide (H₂O₂), superoxide anion (O₂⁻) and peroxynitrite (ONOO) production (μM) were calculated for both Men-treated and untreated samples at different time intervals (24–168 h post-amendment (hpa)) of growth into PDB at 30 °C in dark conditions. Values represent the average of six replicates originated by two biological and three technical replications each ±SE.

Figure 2

PCA score plot of data generated by analysis of several physiological parameters, such as fungal growth, conidiogenesis, aflatoxin B₁ production, catalases and superoxide dismutases activities, hydrogen peroxide, superoxide anion and peroxynitrite production. Observations were clustered according to the sampling time (24–168 hpa) and to the treatment with menadione (Men) or its absence (control).

Figure 3

(A–C). Results of the GO enrichment analysis at 24 (A), 48 (B) and 96 (C) hpa. The x-axis reports the significantly-enriched categories identified among up- (red) and down- (blue) regulated genes after hypergeometric test (p-value < 0.01). The y-axis reports the enrichment score calculated as follows: (number of Differentially Expressed, DE, genes in category X/total number of DE genes)/(number of genes in the genome in category X/total number of genes in the genome).

Figure 3

(A–C). Results of the GO enrichment analysis at 24 (A), 48 (B) and 96 (C) hpa. The x-axis reports the significantly-enriched categories identified among up- (red) and down- (blue) regulated genes after hypergeometric test (p-value < 0.01). The y-axis reports the enrichment score calculated as follows: (number of Differentially Expressed, DE, genes in category X/total number of DE genes)/(number of genes in the genome in category X/total number of genes in the genome).

Figure 4

(A,B) Gene expressions evaluated by the SYBR-green qRT-PCR assay. The housekeeping gene to normalize target gene expression was beta-tubulin (Accession Number AFLA_051840). Relative expression (presented in log2 scale) was calculated by the 2^−ΔΔCt method considering the Men non-treated samples as calibrators in each time interval. The target genes considered were clustered according to their gene ontology, in cell respiration-related genes (aconitase, succinate dehydrogenase, glucose-6-P-dehydrogenase and transaldolase) and in oxidative stress response genes (ap-1 and atfB). Values represent the average of six replicates originated by two biological and three technical replications each ±SE; * p < 0.05; ** p < 0.01 according to the Mann–Whitney test.

Figure 4

(A,B) Gene expressions evaluated by the SYBR-green qRT-PCR assay. The housekeeping gene to normalize target gene expression was beta-tubulin (Accession Number AFLA_051840). Relative expression (presented in log2 scale) was calculated by the 2^−ΔΔCt method considering the Men non-treated samples as calibrators in each time interval. The target genes considered were clustered according to their gene ontology, in cell respiration-related genes (aconitase, succinate dehydrogenase, glucose-6-P-dehydrogenase and transaldolase) and in oxidative stress response genes (ap-1 and atfB). Values represent the average of six replicates originated by two biological and three technical replications each ±SE; * p < 0.05; ** p < 0.01 according to the Mann–Whitney test.

Figure 5

(A,B) Quantification of oxylipins under in vitro experiment. Quantitative LC-MS/MRM analysis of 19 oxylipins (μM) in mycelia of A. flavus treated (menadione) and untreated (control) with Men 0.1 mM, 96 hpa. LOX-oxylipins detected were 9 and 13 hydroxyoctadecatrienoic acid (HOTrE), 9-hydroperoxyoctadecatrienoic acid (HpOTrE), 9-oxo-octadecatrienoic acid (oxoOTrE), 9- and 13-hydroperoxyoctadecadienoic acid (HpODE), 9- and 13-hydroxyoctadecadienoic acid (HODE), 9- and 13-oxo-octadecadienoic acid (oxoODE). LDS-derived oxylipins detected were 8- and 10-hydroxyoctadecadienoic acid (HODE), 8- and 11-hydroperoxyoctadecadienoic acid (HpODE), 9,10- and 12,13-epoxyoctadecenoic acid (epOME), 9,10 and 12,13-dihydroxyoctadecenoic acid (di HOME). Values represent the average of six replicates originated by two biological and three technical replications each ±SE; * p < 0.05; ** p < 0.01 according to the Mann–Whitney test. LOX, lipoxygenases.