The study of fungal species evolved radically with the development of molecular techniques and produced new evidence to understand specific fungal mechanisms such as the production of toxic secondary metabolites. Taking advantage of these technologies to improve food safety, the molecular study of toxinogenic species can help elucidate the mechanisms underlying toxin production and enable the development of new effective strategies to control fungal toxicity. Numerous studies have been made on genes involved in aflatoxin B1 (AFB1) production, one of the most hazardous carcinogenic toxins for humans and animals. The current review presents the roles of these different genes and their possible impact on AFB1 production. We focus on the toxinogenic strains Aspergillus flavus and A. parasiticus, primary contaminants and major producers of AFB1 in crops. However, genetic reports on A. nidulans are also included because of the capacity of this fungus to produce sterigmatocystin, the penultimate stable metabolite during AFB1 production. The aim of this review is to provide a general overview of the AFB1 enzymatic biosynthesis pathway and its link with the genes belonging to the AFB1 cluster. It also aims to illustrate the role of global environmental factors on aflatoxin production and the recent data that demonstrate an interconnection between genes regulated by these environmental signals and aflatoxin biosynthetic pathway.

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Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation.

Aspergillus flavus, a soil-borne pathogen, represents a danger for humans and animals since it produces the carcinogenic mycotoxin Aflatoxin B1 (AFB1). Approaches aiming the reduction of this fungal contaminant mainly involve chemicals that may also be toxic. Therefore, identification and characterization of natural antiaflatoxigenic products represents a sustainable alternative strategy. Piperine, a major component of black and long peppers, has been previously demonstrated as an AFB1-inhibitor; nevertheless its mechanism of action was yet to be elucidated. The aim of the present study was to evaluate piperine's molecular mechanism of action in A. flavus with a special focus on oxidative stress response. For that, the entire AFB1 gene cluster as well as a targeted gene-network coding for fungal stress response factors and cellular receptors were analyzed. In addition to this, fungal enzymatic activities were also characterized. We demonstrated that piperine inhibits aflatoxin production and fungal growth in a dose-dependent manner. Analysis of the gene cluster demonstrated that almost all genes participating in aflatoxin's biosynthetic pathway were down regulated. Exposure to piperine also resulted in decreased transcript levels of the global regulator veA together with an over-expression of genes coding for several basic leucine zipper (bZIP) transcription factors such as atfA, atfB and ap-1 and genes belonging to superoxide dismutase and catalase's families.Furthermore, this gene response was accompanied by a significant enhancement of Version postprintComment citer ce document :

Patulin is the main mycotoxin contaminating apples. During the brewing of alcoholic beverages, this mycotoxin is degraded to ascladiol, which is also the last precursor of patulin. The present study aims (1) to characterize the last step of the patulin biosynthetic pathway and (2) to describe the toxicity of ascladiol. A patE deletion mutant was generated in Penicillium expansum. In contrast to the wild strain, this mutant does not produce patulin but accumulates high levels of E-ascladiol with few traces of Z-ascladiol. This confirms that patE encodes the patulin synthase involved in the conversion of E-ascladiol to patulin. After purification, cytotoxicities of patulin and E- and Z-ascladiol were investigated on human cell lines from liver, kidney, intestine, and immune system. Patulin was cytotoxic for these four cell lines in a dose-dependent manner. By contrast, both E- and Z-ascladiol were devoid of cytotoxicity. Microarray analyses on human intestinal cells treated with patulin and E-ascladiol showed that the latter, unlike patulin, did not alter the whole human transcription. These results demonstrate that E- and Z-ascladiol are not toxic and therefore patulin detoxification strategies leading to the accumulation of ascladiol are good approaches to limit the patulin risk.

Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B1 (AFB1), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant Micromeria graeca—known as hyssop—completely inhibits aflatoxin production by Aspergillus flavus without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of aflR and aflS encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including veA and mtfA, two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as msnA, srrA, catA, cat2, sod1, mnsod, and stuA. In conclusion, hyssop inhibits AFB1 synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB1 contamination.

Patulin (PAT) is a secondary metabolite mainly produced by Aspergillus and Penicillium that is frequently found contaminating apples and rotten fruits. Patulin can be transformed in potencially less toxic compounds such as ascladiol (ASC). Toxic effects of patulin were described in rats and in in vitro models, however concerning ascladiol, data are restricted to metabolic pathways. The aim of the present study was to evaluate the effects of different concentrations of PAT (10 μM, 30 μM, 100 μM) and ASC (30 μM, 100 μM) on intestinal tissue using the jejunal explant model. Explants from pigs were exposed for 4 h to PAT and ASC and after this period were processed for histological, morphometrical and immunohistochemical analysis. Mild histological changes were observed in jejunal explants exposed to PAT and ASC, however no significant difference in the lesional score or villi height was observed between the PAT/ASC-groups and the control. Also, explants exposed to 100 μM of PAT showed a significant decrease in goblet cells density and a significant increase in cell apoptosis. These results indicate that high levels of patulin can induce mild toxic effects on intestinal mucosa whereas ascladiol apparently is non-toxic to intestinal tissue.

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium that contaminate food and feed raw materials. To reduce OTA contamination, we first tested in vitro, actinobacterial strains as potential biocontrol agents and afterward, through a physical decontamination method using activated carbon fibers (ACFs). Actinobacterial strains were screened for their ability to reduce OTA in solid co-culture with A. carbonarius, which is the major OTA-producing species in European vineyards. Four strains showed a high affinity for removing OTA (67%–83%) with no significant effect on fungal growth (<20%). The mechanism of action was first studied by analyzing the expression of OTA cluster genes (acOTApks, acOTAnrps, acOTAhal) by RT-qPCR showing a drastic reduction in all genes (7–15 times). Second, the ability of these strains to degrade OTA was assessed in vitro on ISP2 solid medium supplemented with OTA (100 µg/L). Two strains reduced OTA to undetectable levels. As for the physical method, high adsorption rates were obtained for ACFs at 0.8 g/L with a 50% adsorption of OTA in red wine by AC15 and 52% in grape juice by AC20 within 24 h. These promising methods could be complementarily applied toward reducing OTA contamination in food chains, which promotes food safety and quality.