Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Chitin Deacetylase Homologous Gene cda Contributes to Development and Aflatoxin Synthesis in Aspergillus flavus.

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds.

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance. Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes (stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins. We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

m6A methyltransferase AflIme4 orchestrates mycelial growth, development and aflatoxin B1 biosynthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1), a highly toxic secondary metabolite produced by Aspergillus flavus, poses a severe threat to agricultural production, food safety and human health. The methylation of mRNA m6A has been identified as a regulator of both the growth and AFB1 production of A. flavus. However, its intracellular occurrence and function needs to be elucidated. Here, we identified and characterized a m6A methyltransferase, AflIme4, in A. flavus. The enzyme was localized in the cytoplasm, and knockout of AflIme4 significantly reduced the methylation modification level of mRNA. Compared with the control strains, ΔAflIme4 exhibited diminished growth, conidial formation, mycelial hydrophobicity, sclerotium yield, pathogenicity and increased sensitivity to CR, SDS, NaCl and H2O2. Notably, AFB1 production was markedly inhibited in the A. flavus ΔAflIme4 strain. RNA-Seq coupled with RT-qPCR validation showed that the transcriptional levels of genes involved in the AFB1 biosynthesis pathway including aflA, aflG, aflH, aflK, aflL, aflO, aflS, aflV and aflY were significantly upregulated. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) analysis demonstrated a significant increase in m6A methylation modification levels of these pathway-specific genes, concomitant with a decrease in mRNA stability. These results suggest that AflIme4 attenuates the mRNA stability of genes in AFB1 biosynthesis by enhancing their mRNA m6A methylation modification, leading to impaired AFB1 biosynthesis. Our study identifies a novel m6A methyltransferase AflIme4 and highlights it as a potential target to control A. flavus growth, development and aflatoxin pollution.

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.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Genomic and metabolomic diversity within a familial population of Aspergillus flavus.

Aspergillus flavus is an agriculturally significant micro-fungus having potential to contaminate food and feed crops with toxic secondary metabolites such as aflatoxin (AF) and cyclopiazonic acid (CPA). Research has shown A. flavus strains can overcome heterokaryon incompatibility and undergo meiotic recombination as teleomorphs. Although evidence of recombination in the AF gene cluster has been reported, the impacts of recombination on genotype and metabolomic phenotype in a single generation are lacking. In previous studies, we paired an aflatoxigenic MAT1-1 A. flavus strain with a non-aflatoxigenic MAT1-2 A. flavus strain that had been tagged with green fluorescent protein and then 10 F1 progenies (a mix of fluorescent and non-fluorescent) were randomly selected from single-ascospore colonies and broadly examined for evidence of recombination. In this study, we determined four of those 10 F1 progenies were recombinants because they were not vegetatively compatible with either parent or their siblings, and they exhibited other distinctive traits that could only result from meiotic recombination. The other six progenies examined shared genomic identity with the non-aflatoxigenic, fluorescent, and MAT1-2 parent, but were metabolically distinct. This study highlights phenotypic and genomic changes that may occur in a single generation from the outcrossing of sexually compatible strains of A. flavus.

Response of broilers to dietary inclusion of atoxigenic Aspergillus flavus strain as a biocontrol strategy of aflatoxin.

The objective of this trial was to evaluate how broilers responded to Aspergillus flavus strains that are toxigenic and atoxigenic. The study included four treatments in a 2 × 2 factorial design, with six replicates of 10 birds each. As a result of this study measuring feed intake (FI), weight gain (WG), feed conversion ratio (FCR), crude protein, ether extract, and crude fibre, the interaction was insignificant between the toxigenic and atoxigenic diets (P > 0.05). Consumption of toxigenic aflatoxin B1-500 ppb diet decreased FI and WG but increased FCR, and cost to produce live broiler weight (P < 0.05) compared to the control diets. The addition of atoxigenic strains to contaminated diets significantly offset (P < 0.05) the effects. Diets with or without 500 ppb toxigenic and atoxigenic A. flavus did not affect the relative weight g/100gBW of pancreas, gizzard and bursa of Fabricius. Dietary inclusion of 500 ppb toxigenic Aspergillus spp. increased the relative weight (P < 0.05) of the kidney, liver, spleen and thymus while atoxigenic dietary addition reduced the relative weight of the same organs (P < 0.05). Dietary inclusion of toxigenic and atoxigenic Aspergillus spp. did not significantly affect the haematological parameters measured (P < 0.05). Dietary inclusion of 500 ppb toxigenic Aspergillus elevated the urea, creatine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in the serum of the broilers (P < 0.05). A decrease was observed when atox igenic A. flavus was used in the intervention for urea, creatinine and AST (P < 0.05), whereas an insignificant reduction was observed for ALT and ALP (P ≤ 0.05). This study concluded that dietary atoxigenic strain improved broiler performance, digestibility, and blood parameters.

Liamocins overproduction via the two-pH stage fermentation and anti-Aspergillus flavus activity of Massoia lactone.

Aureobasidium melanogenum was found to be grown the best at the constant pH 7.0 and to produce the highest amount of liamocins at the constant pH 3.0. Therefore, the wild type strain A. melanogenum 9-1 and the engineered strain V33 constructed in the laboratory were grown at the constant pH 7.0 for 48 h, then, they were continued to be cultivated at the constant pH 3.0. Under such conditions, A. melanogenum 9-1 produced 36.51 ± 0.55 g L-1 of liamocin and its cell mass was 27.43 ± 0.63 and 6.00 ± 0.11 g L-1 of glucose was left in the finished medium within 168 h while the engineered strain V33 secreted 70.86 ± 2.04 g L-1 of liamocin, its cell mass was 31.63 ± 0.74 g L-1 , 0.16 ± 0.01 g L-1 of glucose was maintained in the finished medium. Then, Massoia lactone was released from the produced liamocins. The released Massoia lactone loaded in the nanoemulsions could be used to actively damage cell wall and cell membrane of both spores and mycelia of Aspergillus flavus, leading to its cell necrosis. Massoia lactone loaded in the nanoemulsions also actively inhibited cell growth of A. flavus, its conidia production and aflatoxin biosynthesis on peanuts, indicating that Massoia lactone loaded in the nanoemulsions had highly potential application in controlling cell growth of A. flavus and aflatoxin biosynthesis in foods and feedstuffs.

Complexity of fungal polyketide biosynthesis and function.

Where does one draw the line between primary and secondary metabolism? The answer depends on the perspective. Microbial secondary metabolites (SMs) were at first believed not to be very important for the producers because they are dispensable for growth under laboratory conditions. However, such compounds become important in natural niches of the organisms, and some are of prime importance for humanity. Polyketides are an important group of SMs with aflatoxin as a well-known and well-characterized example. In Aspergillus spp., all 34 afl genes encoding the enzymes for aflatoxin biosynthesis are located in close vicinity on chromosome III in a so-called gene cluster. This led to the assumption that most genes required for polyketide biosynthesis are organized in gene clusters. Recent research, however, revealed an enormous complexity of the biosynthesis of different polyketides, ranging from individual polyketide synthases to a gene cluster producing several compounds, or to several clusters with additional genes scattered in the genome for the production of one compound. Research of the last decade furthermore revealed a huge potential for SM biosynthesis hidden in fungal genomes, and methods were developed to wake up such sleeping genes. The analysis of organismic interactions starts to reveal some of the ecological functions of polyketides for the producing fungi.

Expression and application of aflatoxin degrading enzyme gene in Pichia pastoris.

In this study, three aflatoxin degrading enzyme genes, tv-adtz, arm-adtz and cu-adtz, were heterologously expressed in Pichia pastoris. The protein expression of the enzyme solution was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the results showed that specific protein bands were detected and the target genes were successfully integrated into Pichia pastoris. The enzyme activities and detoxification efficiency of TV-ADTZ, Arm-ADTZ and Cu-ADTZ crude enzyme solutions were detected, and the highest enzyme activities were up to 3.57, 4.30, and 2.41 U mL-1 , and the highest degradation rates were up to 45.58%, 60.0% and 34.21%, respectively. Arm-ADTZ with the best degradation effect was selected and designed for detoxification application experiments to test its detoxification efficiency of AFB1 in aqueous phase and in the process of moldy ground corn and preparation of DDGS, respectively, and the degradation rates reached 78.94%, 56.48%, and 24.31% after 24 h of reaction, respectively. Thus, it can be seen that the aflatoxin-degrading enzyme gene was successfully integrated into Pichia pastoris and secreted for expression, and the expressed product could effectively degrade AFB1 .

Role of RNA Modifications, Especially m6A, in Aflatoxin Biosynthesis of Aspergillus flavus.

RNA modifications play key roles in eukaryotes, but the functions in Aspergillus flavus are still unknown. Temperature has been reported previously to be a critical environmental factor that regulates the aflatoxin production of A. flavus, but much remains to be learned about the molecular networks. Here, we demonstrated that 12 kinds of RNA modifications in A. flavus were significantly changed under 29 °C compared to 37 °C incubation; among them, m6A was further verified by a colorimetric method. Then, the transcriptome-wide m6A methylome and m6A-altered genes were comprehensively illuminated through methylated RNA immunoprecipitation sequencing and RNA sequencing, from which 22 differentially methylated and expressed transcripts under 29 °C were screened out. It is especially notable that AFCA_009549, an aflatoxin biosynthetic pathway gene (aflQ), and the m6A methylation of its 332nd adenine in the mRNA significantly affect aflatoxin biosynthesis in A. flavus both on media and crop kernels. The content of sterigmatocystin in both ΔaflQ and aflQA332C strains was significantly higher than that in the WT strain. Together, these findings reveal that RNA modifications are associated with secondary metabolite biosynthesis of A. flavus.

Peppermint essential oil and its nano-emulsion: Potential against aflatoxigenic fungus Aspergillus flavus in food and feed.

A facultative parasite called Aspergillus flavus contaminates several important food crops before and after harvest. In addition, the pathogen that causes aspergillosis infections in humans and animals is opportunistic. Aflatoxin, a secondary metabolite produced by Aspergillus flavus, is also carcinogenic and mutagenic, endangering human and animal health and affecting global food security. Peppermint essential oils and plant-derived natural products have recently shown promise in combating A. flavus infestations and aflatoxin contamination. This review discusses the antifungal and anti-aflatoxigenic properties of peppermint essential oils. It then discusses how peppermint essential oils affect the growth of A. flavus and the biosynthesis of aflatoxins. Several cause physical, chemical, or biochemical changes to the cell wall, cell membrane, mitochondria, and associated metabolic enzymes and genes. Finally, the prospects for using peppermint essential oils and natural plant-derived chemicals to develop novel antifungal agents and protect foods are highlighted. In addition to reducing the risk of aspergillosis infection, this review highlights the significant potential of plant-derived natural products and peppermint essential oils to protect food and feed from aflatoxin contamination and A. flavus infestation.

PP2C phosphatases Ptc1 and Ptc2 dephosphorylate PGK1 to regulate autophagy and aflatoxin synthesis in the pathogenic fungus Aspergillus flavus.

IMPORTANCE: Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus.

Prediction and validation of enzymatic degradation of aflatoxin M1: Genomics and proteomics analysis of Bacillus pumilus E-1-1-1 enzymes.

Aflatoxins are a class of highly toxic mycotoxins. Aflatoxin M1 (AFM1) is hydroxylated metabolite of aflatoxin B1, having comparable toxicity, which is more commonly found in milk. In this study, the whole genome sequencing of Bacillus pumilus E-1-1-1 isolated from feces of 38 kinds of animals, having aflatoxin M1 degradation ability was conducted. Bacterial genome sequencing indicated that a total of 3445 sequences were finally annotated on 23 different cluster of orthologous groups (COG) categories. Then, the potential AFM1 degradation proteins were verified by proteomics; the properties of these proteins were further explored, including protein molecular weight, hydrophobicity, secondary structure prediction, and three-dimensional structures. Bacterial genome sequencing combined with proteomics showed that eight genes were the most capable of degrading AFM1 including three catalases, one superoxide dismutase, and four peroxidases to clone. These eight genes with AFM1 degrading capacity were successfully expressed. These results indicated that AFM1 can be degraded by Bacillus pumilus E-1-1-1 protein and the most degrading proteins were oxidoreductases.

Effect of the aflatoxins B1 and M1 on bovine oocyte developmental competence and embryo morphokinetics.

Aflatoxins are considered as reproductive toxins for mammalian species. Here, we studied the effect of aflatoxin B1 (AFB1) and its metabolite aflatoxin M1 (AFM1) on the development and morphokinetics of bovine embryos. Cumulus oocyte complexes (COCs) were matured with AFB1 (0.032, 0.32, 3.2, 32 µM) or AFM1 (0.015, 0.15, 1.5, 15, 60 nM), then fertilized and the putative zygotes were cultured in an incubator equipped with a time-lapse system. Exposing COCs to 32 µM AFB1 or 60 nM AFM1 reduced the cleavage rate, whereas exposing them to 3.2 or 32 µM AFB1 further reduced the blastocyst formation. A delay was recorded for the first and second cleavages in a dose-dependent manner for both AFB1- and AFM1-treated oocytes. A delay was recorded in the third cleavage in the AFM1-treated group. To explore potential mechanisms, subgroups of COCs were examined for nuclear and cytoplasmic maturation (n = 225; DAPI and FITC-PNA, respectively), and mitochondrial function was examined in a stage-dependent manner. COCs were examined for their oxygen consumption rates (n = 875; Seahorse XFp analyzer) at the end of maturation, MII-stage oocytes were examined for their mitochondrial membrane potential (n = 407; JC1), and putative zygotes were examined using a fluorescent time-lapse system (n = 279; IncuCyte). Exposing COCs to AFB1 (3.2 or 32 µM) impaired oocyte nuclear and cytoplasmic maturation and increased mitochondrial membrane potential in the putative zygotes. These alterations were associated with changes in the expression of mt-ND2 (32 µM AFB1) and STAT3 (all AFM1 concentrations) genes in the blastocyst stage, suggesting a carryover effect from the oocyte to the developing embryos.

Transcriptomic and Metabolomic Analyses of the Response of Resistant Peanut Seeds to Aspergillus flavus Infection.

Peanut seeds are susceptible to Aspergillus flavus infection, which has a severe impact on the peanut industry and human health. However, the molecular mechanism underlying this defense remains poorly understood. The aim of this study was to analyze the changes in differentially expressed genes (DEGs) and differential metabolites during A. flavus infection between Zhonghua 6 and Yuanza 9102 by transcriptomic and metabolomic analysis. A total of 5768 DEGs were detected in the transcriptomic study. Further functional analysis showed that some DEGs were significantly enriched in pectinase catabolism, hydrogen peroxide decomposition and cell wall tissues of resistant varieties at the early stage of infection, while these genes were differentially enriched in the middle and late stages of infection in the nonresponsive variety Yuanza 9102. Some DEGs, such as those encoding transcription factors, disease course-related proteins, peroxidase (POD), chitinase and phenylalanine ammonialyase (PAL), were highly expressed in the infection stage. Metabolomic analysis yielded 349 differential metabolites. Resveratrol, cinnamic acid, coumaric acid, ferulic acid in phenylalanine metabolism and 13S-HPODE in the linolenic acid metabolism pathway play major and active roles in peanut resistance to A. flavus. Combined analysis of the differential metabolites and DEGs showed that they were mainly enriched in phenylpropane metabolism and the linolenic acid metabolism pathway. Transcriptomic and metabolomic analyses further confirmed that peanuts infected with A. flavus activates various defense mechanisms, and the response to A. flavus is more rapid in resistant materials. These results can be used to further elucidate the molecular mechanism of peanut resistance to A. flavus infection and provide directions for early detection of infection and for breeding peanut varieties resistant to aflatoxin contamination.

Aflatoxin B1 Induces Inflammatory Liver Injury via Gut Microbiota in Mice.

Aflatoxin B1 (AFB1), a potent food-borne hepatocarcinogen, is the most toxic aflatoxin that induces liver injury in humans and animals. Species-specific sensitivities of aflatoxins cannot be fully explained by differences in the metabolism of AFB1 between animal species. The gut microbiota are critical in inflammatory liver injury, but it remains to reveal the role of gut microbiota in AFB1-induced liver injury. Here, mice were gavaged with AFB1 for 28 days. Then, the modulation of gut microbiota, colonic barrier, and liver pyroptosis and inflammation were analyzed. To further verify the direct role of gut microbiota in AFB1-induced liver injury, mice were treated with antibiotic mixtures (ABXs) to deplete the microbiota, and fecal microbiota transplantation (FMT) was conducted. The treatment of AFB1 in mice altered gut microbiota composition, such as increasing the relative abundance of Bacteroides, Parabacteroides, and Lactobacillus, inducing colonic barrier dysfunction and promoting liver pyroptosis. In ABX-treated mice, AFB1 had little effect on the colonic barrier and liver pyroptosis. Notably, after FMT, in which the mice were colonized with gut microbiota from AFB1-treated mice, colonic barrier dysfunction, and liver pyroptosis and inflammation were obliviously identified. We proposed that the gut microbiota directly participated in AFB1-induced liver pyroptosis and inflammation. These results provide new insights into the mechanisms of AFB1 hepatotoxicity and pave a window for new targeted interventions to prevent or reduce AFB1 hepatotoxicity.

Aflatoxin B1 Degradation by Ery4 Laccase: From In Vitro to Contaminated Corn.

Aflatoxins (AFs) are toxic secondary metabolites produced by Aspergillus spp. and are found in food and feed as contaminants worldwide. Due to climate change, AFs occurrence is expected to increase also in western Europe. Therefore, to ensure food and feed safety, it is mandatory to develop green technologies for AFs reduction in contaminated matrices. With this regard, enzymatic degradation is an effective and environmentally friendly approach under mild operational conditions and with minor impact on the food and feed matrix. In this work, Ery4 laccase, acetosyringone, ascorbic acid, and dehydroascorbic acid were investigated in vitro, then applied in artificially contaminated corn for AFB1 reduction. AFB1 (0.1 µg/mL) was completely removed in vitro and reduced by 26% in corn. Several degradation products were detected in vitro by UHPLC-HRMS and likely corresponded to AFQ1, epi-AFQ1, AFB1-diol, or AFB1dialehyde, AFB2a, and AFM1. Protein content was not altered by the enzymatic treatment, while slightly higher levels of lipid peroxidation and H2O2 were detected. Although further studies are needed to improve AFB1 reduction and reduce the impact of this treatment in corn, the results of this study are promising and suggest that Ery4 laccase can be effectively applied for the reduction in AFB1 in corn.

A functional intact SUMOylation machinery in Aspergillus flavus contributes to fungal and aflatoxin contamination of food.

SUMO adducts occur in Aspergillus flavus, and are implicated in fungal biology, while the underlying mechanism and the SUMOylation apparatus components in this saprophytic food spoilage mould, remain undefined. Herein, genes encoding SUMOylation cascade enzymes in A. flavus, including two heterodimeric SUMO E1 activating enzymes, a unique SUMO E2 conjugating enzyme, and one of SUMO E3 ligases, were identified and functionally analyzed. Global SUMO adducts immunoassay, multiple morphological comparison, aflatoxin attributes test, fungal infection and transcriptomic analyses collectively revealed that: E1 and E2 were essential for intracellular SUMOylation, and contributed to both stress response and fungal virulence-related events, including sporulation, colonization, aflatoxins biosynthesis; the primary E3 in this fungus, AfSizA, might serve as the molecular linkage of SUMOylation pathway to fungal virulence rather than SUMOylation-mediated stress adaptation. These findings demonstrated that SUMOylation machinery in A. flavus was functionally intact and contributed to multiple pathobiological processes, hence offering ideas and targets to control food contamination by this mycotoxigenic fungus.