Fus3 Interacts with Gal83, Revealing the MAPK Crosstalk to Snf1/AMPK to Regulate Secondary Metabolic Substrates in Aspergillus flavus.

Aflatoxins (AFs), highly carcinogenic natural products, are produced by the secondary metabolism of fungi such as Aspergillus flavus. Essential for the fungi to respond to environmental changes and aflatoxin synthesis, the pheromone mitogen-activated protein kinase (MAPK) is a potential regulator of aflatoxin biosynthesis. However, the mechanism by which pheromone MAPK regulates aflatoxin biosynthesis is not clear. Here, we showed Gal83, a new target of Fus3, and identified the pheromone Fus3-MAPK signaling pathway as a regulator of the Snf1/AMPK energy-sensing pathway modulating aflatoxins synthesis substrates. The screening for Fus3 target proteins identified the ß subunit of Snf1/AMPK complexes using tandem affinity purification and multiomics. This subunit physically interacted with Fus3 both in vivo and in vitro and received phosphorylation from Fus3. Although the transcript levels of aflatoxin synthesis genes were not noticeably downregulated in both gal83 and fus3 deletion mutant strains, the levels of aflatoxin B1 and its synthesis substrates and gene expression levels of primary metabolizing enzymes were significantly reduced. This suggests that both the Fus3-MAPK and Snf1/AMPK pathways respond to energy signals. In conclusion, all the evidence unlocks a novel pathway of Fus3-MAPK to regulate AFs synthesis substrates by cross-talking with the Snf1/AMPK complexes.

Identification of a Novel Bacillus velezensis IS-6 Nudix Hydrolase Nh-9 Involved in Ochratoxin A Detoxification by Transcriptomic Profiling and Functional Verification.

Contamination of foods and feeds with Ochratoxin A (OTA) is a global problem, and its detoxification is challenging. In this study, Bacillus velezensis IS-6 culture isolate supernatant degraded 1.5 g/mL OTA by 89% after 24 h of incubation at 37 °C, whereas viable cells and intra-cell extracts were less effective. The OTA degradation by B. velezensis IS-6 was an enzymatic process mediated by the culture supernatant. The degradation activity was optimal at 37 °C and pH 7.0, and Fe2+ and Cu2+ ions enhanced the OTA degradation. The LC-MS/MS analysis confirmed that structure of OTA was modified, resulting in the production of OTα that was less toxic than OTA. The transcriptomic analysis of B. velezensis IS-6 showed that 38 differentially expressed genes (DEGs) were significantly up-regulated, and 24 DEGs were down-regulated after treatment with OTA. A novel OTA degradation enzyme Nudix hydrolase Nh-9 was successfully cloned and characterized from the up-regulated genes. The recombinant Nh-9 enzyme was overexpressed in Escherichia coli BL21 and purified by affinity chromatography, exhibiting 68% degradation activity against 1.0 µg/mL OTA at 37 °C in 24 h. The degraded product by the Nh-9 enzyme was identified as the less toxic OTα by LC-MS/MS. According to the findings, it can be inferred that Nh-9 is the main OTA-degrading enzyme in B. velezensis IS-6. Furthermore, OTA may be co-degraded by Nh-9, carboxylesterase, signal peptidase, and other degrading agents that are yet to be discovered in this strain.

Corepressors SsnF and RcoA Regulate Development and Aflatoxin B1 Biosynthesis in Aspergillus flavus NRRL 3357.

Aspergillus flavus is a saprophytic fungus that can be found across the entire world. It can produce aflatoxin B1 (AFB1), which threatens human health. CreA, as the central factor in carbon catabolite repression (CCR), regulates carbon catabolism and AFB1 biosynthesis in A. flavus. Additionally, SsnF-RcoA are recognized as the corepressors of CreA in CCR. In this study, ssnF and rcoA not only regulated the expressions of CCR factors and hydrolase genes, but also positively affected mycelia growth, conidia production, sclerotia formation, and osmotic stress response in A. flavus. More importantly, SsnF and RcoA were identified as positive regulators for AFB1 biosynthesis, as they modulate the AF cluster genes and the relevant regulators at a transcriptional level. Additionally, the interactions of SsnF-CreA and RcoA-CreA were strong and moderate, respectively. However, the interaction of SsnF and RcoA was weak. The interaction models of CreA-SsnF, CreA-RcoA, and SsnF-RcoA were also simulated with a docking analysis. All things considered, SsnF and RcoA are not just the critical regulators of the CCR pathway, but the global regulators involving in morphological development and AFB1 biosynthesis in A. flavus.

Fus3, as a Critical Kinase in MAPK Cascade, Regulates Aflatoxin Biosynthesis by Controlling the Substrate Supply in Aspergillus flavus, Rather than the Cluster Genes Modulation.

The Fus3-MAP kinase module is a conserved phosphorylation signal system in eukaryotes that responds to environmental stress and transduction of external signals from the outer membrane to the nucleus. Aspergillus flavus can produce aflatoxins (AF), which seriously threaten human and animal health. In this study, we determined the functions of Fus3, confirmed Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylation, and explored the possible phosphorylation motifs and potential targets of Fus3. The regulatory mechanism of Fus3 on the biosynthesis of AF was partly revealed in this study. AF production was downregulated in Δfus3, but the transcriptional expression of most AF cluster genes was upregulated. It is notable that the levels of acetyl-CoA and malonyl-CoA, the substrates of AF, were significantly decreased in fus3 defective strains. Genes involved in acetyl-CoA and malonyl-CoA biosynthesis were significantly downregulated at transcriptional or phosphorylation levels. Specifically, AccA might be a direct target of Fus3, which led to acetyl-CoA carboxylase activities were decreased in null-deletion and site mutagenesis strains. The results concluded that Fus3 could regulate the expression of acetyl-CoA and malonyl-CoA biosynthetic genes directly or indirectly, and then affect the AF production that relies on the regulation of AF substrate rather than the modulation of AF cluster genes. IMPORTANCE Aspergillus flavus is an important saprophytic fungus that produces aflatoxins (AF), which threaten food and feed safety. MAP (mitogen-activated protein) kanases are essential for fungal adaptation to diverse environments. Fus3, as the terminal kinase of a MAPK cascade, interacts with other MAPK modules and phosphorylates downstream targets. We provide evidence that Fus3 could affect AF biosynthesis by regulating the production of acetyl-CoA and malonyl-CoA, but this does not depend on the regulation of AF biosynthetic genes. Our results partly reveal the regulatory mechanism of Fus3 on AF biosynthesis and provide a novel AF modulation pattern, which may contribute to the discovery of new strategies in controlling A. flavus and AF contamination.

The Regulatory Mechanism of Water Activities on Aflatoxins Biosynthesis and Conidia Development, and Transcription Factor AtfB Is Involved in This Regulation.

Peanuts are frequently infected by Aspergillus strains and then contaminated by aflatoxins (AF), which brings out economic losses and health risks. AF production is affected by diverse environmental factors, especially water activity (aw). In this study, A. flavus was inoculated into peanuts with different aw (0.90, 0.95, and 0.99). Both AFB1 yield and conidia production showed the highest level in aw 0.90 treatment. Transcriptional level analyses indicated that AF biosynthesis genes, especially the middle- and later-stage genes, were significantly up-regulated in aw 0.90 than aw 0.95 and 0.99. AtfB could be the pivotal regulator response to aw variations, and could further regulate downstream genes, especially AF biosynthesis genes. The expressions of conidia genes and relevant regulators were also more up-regulated at aw 0.90 than aw 0.95 and 0.99, suggesting that the relative lower aw could increase A. flavus conidia development. Furthermore, transcription factors involved in sexual development and nitrogen metabolism were also modulated by different aw. This research partly clarified the regulatory mechanism of aw on AF biosynthesis and A. flavus development and it would supply some advice for AF prevention in food storage.

Comprehensive Transcriptome and Proteome Analyses Reveal the Modulation of Aflatoxin Production by Aspergillus flavus on Different Crop Substrates.

As a natural severe contaminant of stored grains and other crops worldwide, Aspergillus flavus can produce aflatoxins (AFs), the most powerful naturally producing toxic and hepatocarcinogenic compounds. AFs production is regulated by diverse factors including AFs cluster genes, transcription factors, regulators, and environmental factors. Among them, crop substrate is one of the most important factors. Here, we found that AFB1 production was significantly higher in maize and rice broth than in peanut broth. To clarify the mechanisms involved, complementary transcriptomic and proteomic analyses were performed to identify changes in A. flavus incubated in the three crop substrates. The results indicated that fewer genes and proteins were differentially expressed between maize and rice substrates, whereas more differentially expressed genes were observed between maize/rice broth and peanut broth. In particular, the genes involved in the initial step of AFs biosynthesis (aflA, aflB, and aflC) and the ACCase-encoding gene accA were significantly upregulated on the maize and rice substrates. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses indicated that carbon-metabolism-related genes were obviously enriched in the maize broth, and the genes involved in acetyl-CoA accumulation and consumption were up- and downregulated, respectively. Several genes involved in the regulation of AFs biosynthesis, including veA, ppoB, snf1, and the G-protein-coupled receptor (GPCR) genes, were differentially expressed on the three substrates, suggesting that these genes may be also involved in sugar signal sensing, transfer, and regulation. Interestingly, by the correlation analyses of transcriptome and proteome, trehalose metabolism genes, aldehyde dehydrogenase gene, and tryptophan synthase gene were found to be relevant with the regulation of AFs production on different crop substrates. Taken together, the differential expressions of the AFs cluster genes, several regulatory genes, and carbon metabolism genes were involved in the comprehensive modulation of AFs production on different crop substrates.

The Complex Essential Oils Highly Control the Toxigenic Fungal Microbiome and Major Mycotoxins During Storage of Maize.

The contamination of maize with fungi and subsequent mycotoxins is a pivotal and long-standing safety concern in the maize industry. In this study, the inhibitory effects of the complex essential oils (cinnamaldehyde, citral, eugenol, and menthol, 3:3:2:2, v/v) on fungal growth and mycotoxins production in stored maize were evaluated using traditional plate counting, internal transcribed spacer 2 (ITS2) sequencing and liquid chromatography-tandem mass spectrometry. Complex essential oils (0.02%) significantly (p < 0.05) reduced the total fungi counts and the content of aflatoxin B1, zearalenone, and deoxynivalenol in stored maize during 12 months of storage, and were more effective than propionic acid (0.2%). The fungal diversity of the control group was the highest with 113 operational taxonomic units. During storage of maize kernels, Aspergillus, Fusarium, Wallemia, Sarocladium, and Penicillium were main genera. At 0-6 months, the fungal diversity was high and Fusarium was predominant genus. However, at 7-11 months, the fungal diversity was low and Aspergillus was predominant genus. During the later stages of storage, the prevalence of Aspergillus in maize treated with essential oils was significantly lower than (p < 0.05) that observed in the propionic acid treated and control samples. The results of this study suggest that the complex essential oils may be employed successfully to control toxigenic fungi and subsequent contamination with mycotoxins in maize.

The anti-aflatoxigenic mechanism of cinnamaldehyde in Aspergillus flavus.

Aflatoxin B1 (AFB1), the predominant and most carcinogenic naturally polyketide, is mainly produced by Aspergillus flavus and Aspergillus parasiticus. Cinnamaldehyde has been reported for inhibiting the growth and aflatoxin biosynthesis in A. flavus. But its molecular mechanism of action still remains largely ambiguous. Here, the anti-aflatoxigenic mechanism of cinnamaldehyde in A. flavus was investigated via a comparative transcriptomic analysis. The results indicated that twenty five of thirty genes in aflatoxin cluster showed down-regulation by cinnamaldehyde although the cluster regulators aflR and aflS were slightly up-regulated. This may be due to the up-regulation of the oxidative stress-related genes srrA, msnA and atfB being caused by the significant down-regulation of the diffusible factor FluG. Cinnamaldehyde also inhibited aflatoxin formation by perturbing GPCRs and oxylipins normal function, cell wall biosynthesis and redox equilibrium. In addition, accumulation of NADPH due to up-regulation of pentose phosphate pathway drove acetyl-CoA to lipids synthesis rather than polyketides. Both GO and KEGG analysis suggested that pyruvate and phenylalanine metabolism, post-transcriptional modification and key enzymes biosynthesis might be involved in the suppression of AFB1 production by cinnamaldehyde. This study served to decipher the anti-aflatoxigenic properties of cinnamaldehyde in A. flavus and provided powerful evidence for its use in practice.

Large-Scale Comparative Analysis of Eugenol-Induced/Repressed Genes Expression in Aspergillus flavus Using RNA-seq.

Aflatoxin B1 (AFB1), which is mainly produced by Aspergillus flavus and Aspergillus parasiticus, is the most toxic and hepatocarcinogenic polyketide known. Chemical fungicides are currently utilized to reduce this fungal contaminant, but they are potentially harmful to human health and the environment. Therefore, natural anti-aflatoxigenic products are used as sustainable alternatives to control food and feed contamination. For example, eugenol, presents in many essential oils, has been identified as an aflatoxin inhibitor. However, its exact mechanism of inhibition is yet to be clarified. In this study, the anti-aflatoxigenic mechanism of eugenol in A. flavus was determined using a comparative transcriptomic approach. Twenty of twenty-nine genes in the aflatoxin biosynthetic pathway were down-regulated by eugenol. The most strongly down-regulated gene was aflMa, followed by aflI, aflJ, aflCa, aflH, aflNa, aflE, aflG, aflM, aflD, and aflP. However, the expression of the regulator gene aflR did not change significantly and the expression of aflS was slightly up-regulated. The down-regulation of the global regulator gene veA resulted in the up-regulation of srrA, and the down-regulation of ap-1 and mtfA. The early developmental regulator brlA was profoundly up-regulated in A. flavus after eugenol treatment. These results suggested a model in which eugenol improves fungal development by up-regulating the expression of brlA by the suppression of veA expression and inhibits aflatoxin production through the suppression of veA expression. Exposure to eugenol also caused dysregulated transcript levels of the G protein-coupled receptors (GPCRs) and oxylipins genes. A Gene Ontology analysis indicated that the genes that were highly responsive to eugenol were mainly enriched in RNA-binding functions, suggesting that post-transcriptional modification plays a pivotal role in aflatoxin biosynthesis. KEGG analysis showed that ribosome biogenesis was the most dysregulated pathway, suggesting that eugenol dysregulates ribosome biogenesis, which then interrupts the biosynthesis of Nor-1, Ver-1, and OmtA, and prevents aflatoxisomes performing their normal function in aflatoxin production. In conclusion, our results indicated that eugenol inhibited AFB1 production by modulating the expression of structural genes in aflatoxin pathway, fungal antioxidant status, post-transcriptional modifications and biosynthesis of backbone enzymes in A. flavus.