Interindividual Differences in In Vitro Human Intestinal Microbial Conversion of 3-Acetyl-DON and 15-Acetyl-DON

In order to evaluate the potential differences between 3-Ac-DON and 15-Ac-DON in the human intestinal microbial metabolism, human fecal samples were anaerobically cultured in vitro. Quantitative fecal microbiota characteristics were obtained by 16S rRNA sequencing, and the data revealed several genera that may be relevant for the transformation of the acetylated DONs. Significant differences in the level of 3-Ac-DON and 15-Ac-DON conversion were observed among microbiota from different human individuals. 3-Ac-DON could be rapidly hydrolyzed; a ten-fold difference was observed between the highest and lowest in vitro conversion after 4 h. However, 15-Ac-DON was not fully transformed in the 4 h culture of all the individual samples. In all cases, the conversion rate of 3-Ac-DON was higher than that of 15-Ac-DON, and the conversion rate of 3-Ac-DON into DON varied from 1.3-to 8.4-fold that of 15-Ac-DON. Based on in vitro conversion rates, it was estimated that 45–452 min is required to convert all 3-Ac-DON to DON, implying that deacetylation of 3-Ac-DON is likely to occur completely in all human individuals during intestinal transit. However, for conversion of 15-Ac-DON, DON formation was undetectable at 4 h incubation in 8 out of the 25 human samples, while for 7 of these 8 samples conversion to DON was detected at 24 h incubation. The conversion rates obtained for these seven samples indicated that it would take 1925–4805 min to convert all 15-Ac-DON to DON, while the other 17 samples required 173–734 min. From these results it followed that for eight of the 25 individuals, conversion of 15-Ac-DON to DON was estimated to be incomplete during the 1848 min intestinal transit time. The results thus indicate substantial interindividual as well as compound specific differences in the deconjugation of acetylated DONs. A spearman correlation analysis showed a statistically significant relationship between deconjugation of both acetyl-DONs at 4 h and 24 h incubation. Based on the in vitro kinetic parameters and their scaling to the in vivo situation, it was concluded that for a substantial number of human individuals the deconjugation of 15-Ac-DON may not be complete upon intestinal transit

Dimethylformamide Inhibits Fungal Growth and Aflatoxin B1 Biosynthesis in Aspergillus flavus by Down-Regulating Glucose Metabolism and Amino Acid Biosynthesis

Aflatoxins (AFs) are secondary metabolites produced by plant fungal pathogens infecting crops with strong carcinogenic and mutagenic properties. Dimethylformamide (DMF) is an excellent solvent widely used in biology, medicine and other fields. However, the effect and mechanism of DMF as a common organic solvent against fungal growth and AFs production are not clear. Here, we discovered that DMF had obvious inhibitory effect against A. flavus, as well as displayed complete strong capacity to combat AFs production. Hereafter, the inhibition mechanism of DMF act on AFs production was revealed by the transcriptional expression analysis of genes referred to AFs biosynthesis. With 1% DMF treatment, two positive regulatory genes of AFs biosynthetic pathway aflS and aflR were down-regulated, leading to the suppression of the structural genes in AFs cluster like aflW, aflP. These changes may be due to the suppression of VeA and the subsequent up-regulation of FluG. Exposure to DMF caused the damage of cell wall and the dysfunction of mitochondria. In particular, it is worth noting that most amino acid biosynthesis and glucose metabolism pathway were down-regulated by 1% DMF using Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Taken together, these RNA-Seq data strongly suggest that DMF inhibits fungal growth and aflatoxin B1 (AFB1) production by A. flavus via the synergistic interference of glucose metabolism, amino acid biosynthesis and oxidative phosphorylation

Edwardsiella tarda Outer Membrane Protein C: An Immunogenic Protein Induces Highly Protective Effects in Flounder (Paralichthys olivaceus) against Edwardsiellosis

Outer membrane protein C of Edwardsiella tarda is a major cell surface antigen and it was identified to be an immunogenic protein by Western blot using flounder (Paralichthys olivaceus) anti-recombinant OmpC (rOmpC), and anti-E. tarda antibodies. rOmpC tested the immune protective effect against E. tarda challenge in a flounder model and produced a relative percentage of survival rate of 85%. The immune response of flounder induced by rOmpC was investigated, and the results showed that: (1) the levels of specific serum antibodies induced by rOmpC were significantly higher than the control group after the second week after immunization, and the peak level occurred at week five after immunization; (2) rOmpC could induce the proliferation of sIg+ lymphocytes, and the peak levels of sIg+ lymphocytes in blood, spleen, and pronephros occurred at 4–5 weeks after immunization; and (3) the MHCIIα, CD4-1, IL-1β, IL-6 and TNF-α genes were significantly induced after being injected with rOmpC. Taken together, these results demonstrated that rOmpC could evoke highly protective effects against E. tarda challenge and induce strong innate immune response and humoral immune response of flounder, which indicated that OmpC was a promising vaccine candidate against E. tarda infection

Metagenome Analysis Identifies Microbial Shifts upon Deoxynivalenol Exposure and Post-Exposure Recovery in the Mouse Gut

Deoxynivalenol (DON) is one of the most prevalent food-associated mycotoxins, and is known to cause a variety of adverse health effects on human and animals. Upon oral exposure, the intestine is the main target organ of DON. The current study unraveled that DON exposure (2 mg/kg bw/day or 5 mg/kg bw/day) can significantly reshape the gut microbiota in a mouse model. The study characterized the specific gut microbial strains and genes changed after DON exposure and also investigated the recovery of the microbiota upon either 2 weeks daily prebiotic inulin administration or 2 weeks recovery without intervention after termination of DON exposure (spontaneous recovery). The results obtained reveal that DON exposure causes a shift in gut microorganisms, increasing the relative abundance of Akkermansia muciniphila, Bacteroides vulgatus, Hungatella hathewayi, and Lachnospiraceae bacterium 28-4, while the relative abundance of Mucispirillum schaedleri, Pseudoflavonifractor sp. An85, Faecalibacterium prausnitzii, Firmicutes bacterium ASF500, Flavonifractor plautii, Oscillibacter sp. 1-3, and uncultured Flavonifractor sp. decreased. Notably, DON exposure enhanced the prevalence of A. muciniphila, a species considered as a potential prebiotic in previous studies. Most of the gut microbiome altered by DON in the low- and high-dose exposure groups recovered after 2 weeks of spontaneous recovery. Inulin administration appeared to promote the recovery of the gut microbiome and functional genes after low-dose DON exposure, but not after high-dose exposure, at which changes were exacerbated by inulin-supplemented recovery. The results obtained help to better understand the effect of DON on the gut microbiome, and the gut microbiota’s recovery upon termination of DON exposure

Interaction between food-borne mycotoxins and gut microbiota : A review

Contamination of food and feed by mycotoxins is considered one of most serious food safety problems in the world, because these fungal metabolites can be teratogenic, mutagenic, carcinogenic and immunosuppressive, and can cause serious damages to animal and human health. Mycotoxins may modulate the gut microbiota with potential consequences for gut and host health. On the other hand, the gut microbiota may metabolize the mycotoxins thereby converting them to a form with different activity. Chemical-microbial interactions can be categorized into two classes: Microbiome Modulation of Toxicity (MMT) and Toxicant Modulation of the Microbiome (TMM). The present review provides a state-of-the-art overview of this bi-directional interaction between the major food-borne mycotoxins such as aflatoxins, ochratoxins, deoxynivalenols and zearalenone present in food, feed and the gut microbiota. In addition, the effect of probiotics on gut microbiota in animals exposed to mycotoxins is summarized. Possible consequences of the role of gut microbiota for the risk assessment of mycotoxins, are also discussed. It is concluded that without taking the role of the gut microbiota into account effects of food-borne mycotoxins on health may be underestimated.</p

Genome-Wide Identification and Expression Analysis of the Basic Leucine Zipper (bZIP) Transcription Factor Gene Family in <i>Fusarium graminearum</i>

The basic leucine zipper (bZIP) is a widely found transcription factor family that plays regulatory roles in a variety of cellular processes including cell growth and development and various stress responses. However, the bZIP gene family has not been well studied at a genome-wide scale in Fusarium graminearum (Fg), a potent pathogen of cereal grains. In the present study, we conducted a genome-wide identification, characterization, and expression profiling of 22 F. graminearum bZIP (FgbZIP) genes at different developmental stages and under various abiotic stresses. All identified FgbZIPs were categorized into nine groups based on their sequence similarity and phylogenetic tree analysis. Furthermore, the gene structure analysis, conserved motif analysis, chromosomal localization, protein network studies, and synteny analysis were performed. The symmetry of the exon and intron varied with the phylogenetic groups. The post-translational modifications (PTMs) analysis also predicted several phosphorylation sites in FgbZIPs, indicating their functional diversity in cellular processes. The evolutionary study identified many orthogroups among eight species and also predicted several gene duplication events in F. graminearum. The protein modeling indicated the presence of a higher number of α-helices and random coils in their structures. The expression patterns of FgbZIP genes showed that 5 FgbZIP genes, including FgbZIP_1.1, FgbZIP_1.3, FgbZIP_2.6 FgbZIP_3.1 and FgbZIP_4.3, had high expression at different growth and conidiogenesis stages. Similarly, eight genes including FgbZIP_1.1, FgbZIP_1.6, FgbZIP_2.3, FgbZIP_2.4, FgbZIP_4.1, FgbZIP_4.2, FgbZIP_4.3 and FgbZIP_4.6 demonstrated their putative role in response to various abiotic stresses. In summary, these results provided basic information regarding FgbZIPs which are helpful for further functional analysis

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

A Novel and Label-Free Chemiluminescence Detection of Zearalenone Based on a Truncated Aptamer Conjugated with a G-Quadruplex DNAzyme

Zearalenone (ZEN), one of the most frequently occurring mycotoxin contaminants in foods and feeds, poses considerable threat to human and animal health, owing to its acute and chronic toxicities. Thus, rapid and accurate detection of ZEN has attracted broad research interest. In this work, a novel and label-free chemiluminescence aptasensor based on a ZEN aptamer and a G-quadruplex DNAzyme was constructed. It was established on a competitive assay between ZEN and an auxiliary DNA for the aptamer, leading to activation of the G-quadruplex/hemin DNAzyme and subsequent signal amplification by chemiluminescence generation after substrate addition. To maximize the detection sensitivity, numerous key parameters including truncated aptamers were optimized with molecular docking analysis. Upon optimization, our aptasensor exhibited a perfect linear relationship (R2 = 0.9996) for ZEN detection in a concentration range of 1–100 ng/mL (3.14–314.10 nM) within 40 min, achieving a detection limit of 2.85 ng/mL (8.95 nM), which was a 6.7-fold improvement over that before optimization. Most importantly, the aptasensor obtained a satisfactory recovery rate of 92.84–137.27% and 84.90–124.24% for ZEN-spiked wheat and maize samples, respectively. Overall, our label-free chemiluminescence aptasensor displayed simplicity, sensitivity, specificity and practicality in real samples, indicating high application prospects in the food supply chain for rapid detection of ZEN

Species Differences in in vitro and Estimated in vivo Kinetics for Intestinal Microbiota Mediated Metabolism of Acetyl-deoxynivalenols

Scope: Deoxynivalenol (DON) and its acetylated derivatives 3-acetyl-DON (3-Ac-DON) and 15-acetyl-DON (15-Ac-DON) are important mycotoxins of concern in the modern food chain. Methods and Results: The present study reveals that the rate of de-acetylation in in vitro anaerobic fecal incubations decreased in the order rat > mouse > human > pig for 3-Ac-DON, and mouse > human > rat > pig for 15-Ac-DON. The ratio between the de-acetylation rate of 3-Ac-DON and 15-Ac-DON varies with the species. Scaling of the kinetic parameters to the in vivo situation results in catalytic efficiencies decreasing in the order human > rat > pig > mouse for 3-Ac-DON and human > pig > rat > mouse for 15-Ac-DON. The results obtained indicate that in mice, 3-Ac-DON can be fully deconjugated while 15-Ac-DON cannot. In rats, pigs, and humans, both 3-Ac-DON and 15-Ac-DON can be totally transformed by gut fecal microbiota during the estimated intestinal residence time. A correlation analysis between the deacetylation rate and the relative abundance of the microbiome suggests Lachnospiraceae may be involved in the deacetylation process. Conclusion: It is concluded that interspecies differences in deacetylation of acetylated DONs exist but that in risk assessment assumption of complete intestinal deconjugation provides an adequate approach.</p

Corepressors SsnF and RcoA Regulate Development and Aflatoxin B₁ Biosynthesis in <i>Aspergillus flavus</i> 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