Modulation of fungal toxin production by natural extracts

Mycotoxin’s contamination represents an important source of food spoilage that has to be taken in consideration. For years pesticides, have been used as a common strategy to combat mycotoxin contamination. However, such products were also demonstrated to be harmful to humans and animals’ health. Therefore, it is necessary to find new strategies in order to avoid mycotoxin contamination and the use of natural products could be a promising alternative. Indeed, these compounds may be eco-friendly and several of them are demonstrated aseffective agents against toxin production. Nevertheless, their precise mechanism of action is poorly documented. One of the principal aim of this work consisted in the identification of natural sources capable to inhibit mycotoxin production and in the elucidation of their molecular mechanism of action. For that, we developed a molecular tool aiming the analysis of the impact of natural extracts on the expression of several genes related to Aflatoxin B1 synthesis in Aspergillus flavus. The study of this mycotoxin is an important issue since it is one of the most dangerous compounds inducing cancer in humans and animals. Taking advantage of the well-studied genome of Aspergillus flavus and considering that AFB1 production involves a great number of genetic elements, a q-PCR approach including 60 of the principal genes involved in toxin biosynthesis was developed. This tool simultaneously studies the entire AFB1 gene cluster but also 33 regulatory factors coding for external stimuli to which fungus is exposed. Using this molecular approach, the study of already known but also, new sources of anti-aflatoxigenic compounds was performed. The molecular mechanism of action of 2 isolated molecules and 3 whole plant extracts were determined and the impact of these compounds on the genes involved in AFB1 production was analysed. One of the innovative findings consisted in the demonstration that natural products systematically modulate several genes within AFB1’s inhibition. Taken together, our approach demonstrated that the use of natural products against mycotoxin production can represent an alternative strategy to inhibit food contamination. The elucidation of the mechanism of action of natural products allowed a better understanding of the fungal machinery through which toxin can be inhibited

Streptomyces roseolus, A Promising Biocontrol Agent Against Aspergillus flavus, the Main Aflatoxin B1 Producer

Crop contamination by aflatoxin B1 is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute to the substitution of phytopharmaceutical products are thus needed. For this, a promising method resides in the use of biocontrol agents. Several actinobacteria strains have demonstrated to effectively reduce the aflatoxin B1 concentration. Nevertheless, the molecular mechanism of action by which these biological agents reduce the mycotoxin concentration has not been determined. The aim of the present study was to test the potential use of Streptomyces roseolus as a biocontrol agent against aflatoxin B1 contamination. Co-cultures with Aspergillus flavus were conducted, and the molecular fungal response was investigated through analyzing the q-PCR expression of 65 genes encoding relevant fungal functions. Moreover, kojic and cyclopiazonic acid concentrations, as well as morphological fungal changes were also analyzed. The results demonstrated that reduced concentrations of aflatoxin B1 and kojic acid were respectively correlated with the down-regulation of the aflatoxin B1 gene cluster and kojR gene expression. Moreover, a fungal hypersporulated phenotype and a general over-expression of genes involved in fungal development were observed in the co-culture conditio

Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

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

Influence of the culture conditions on the production of NGPs by Aspergillus tubingensis

The filamentous fungus Aspergillus tubingensis that belongs to the black Aspergillus section has the capacity to produce high-value metabolites, for instance, Naphtho-Gamma-Pyrones (NGPs). For these fungal secondary metabolites, numerous biological properties of industrial interest have been demonstrated, such as antimicrobial, antioxidant and anti-cancer capacities. It has been observed that these secondary metabolites production is linked with the fungal sporulation. The aim of this research was to apply environmental stresses to trigger the production of NGPs in liquid cultures with CYB (Czapek Dox Broth): osmotic and oxidative stresses. In addition, numerous parameters were tested during the experiments, such as pH value, incubation time, container geometry, and static and agitation conditions. Results demonstrate that the produced amount of NGPs can be enhanced by decreasing the water activity (aw) or by adding an oxidative stress factor. In conclusion, this study can contribute to our knowledge regarding A. tubingensis to present an effective method to increase NGPs's production, which may support the development of current industrial processes

Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

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

Streptomyces roseolus, A Promising Biocontrol Agent Against Aspergillus flavus, the Main Aflatoxin B1 Producer

Crop contamination by aflatoxin B1 is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute to the substitution of phytopharmaceutical products are thus needed. For this, a promising method resides in the use of biocontrol agents. Several actinobacteria strains have demonstrated to effectively reduce the aflatoxin B1 concentration. Nevertheless, the molecular mechanism of action by which these biological agents reduce the mycotoxin concentration has not been determined. The aim of the present study was to test the potential use of Streptomyces roseolus as a biocontrol agent against aflatoxin B1 contamination. Co-cultures with Aspergillus flavus were conducted, and the molecular fungal response was investigated through analyzing the q-PCR expression of 65 genes encoding relevant fungal functions. Moreover, kojic and cyclopiazonic acid concentrations, as well as morphological fungal changes were also analyzed. The results demonstrated that reduced concentrations of aflatoxin B1 and kojic acid were respectively correlated with the down-regulation of the aflatoxin B1 gene cluster and kojR gene expression. Moreover, a fungal hypersporulated phenotype and a general over-expression of genes involved in fungal development were observed in the co-culture condition

Modulation de la biosynthèse de l'aflatoxine B1 chez Aspergillus flavus par des substances et extraits végétaux

La contamination des aliments par les mycotoxines est une source très importante de gaspillage de nourriture. Depuis des années, la stratégie classique pour lutter contre ces contaminants est l’utilisation de pesticides. Cependant, il a été montré que ces produits pouvaient avoir des effets nocifs sur la biodiversité et la santé humaine et animale. Il est donc nécessaire de développer de nouvelles stratégies, plus écologiques, pour lutter contre les mycotoxines. L’utilisation de produits naturels pourrait représenter une alternative intéressante et plus respectueuse de l’environnement. En effet, certains produits naturels sont capables d’inhiber la production de mycotoxines. Cependant, le mécanisme d’action mis en jeu est souvent mal compris. Un des objectifs principaux de ce travail a consisté à identifier des produits naturels capables d’inhiber la production de mycotoxines et d’élucider leur mécanisme d’action moléculaire. Pour ce faire, un outil moléculaire a été conçu pour comprendre les mécanismes permettant à des produits naturels d’inhiber la production d’Aflatoxine B1 (AFB1) chez Aspergillus flavus. L’étude de cette mycotoxine est importante puisque c’est un cancérigène puissant chez l’homme et les animaux. Un outil moléculaire permettant l’analyse simultanée de l’expression de 60 des principaux gènes impliqués dans la synthèse de l’aflatoxine B1 par q-*-PCR a été développé. Il permet d’étudier l’ensemble des gènes du cluster d’AFB1 mais également 33 autres gènes codant pour des facteurs de régulation liés à l’environnement dans lequel se trouve la moisissure. Par cette approche, le mécanisme d'action moléculaire de l’eugénol et la pipérine, ainsi que celui de 3 extraits naturels de plantes a été identifié et l'impact de ces composés sur les gènes impliqués dans la production d'AFB1 a été élucidé. L'un des principaux résultats de cette étude a été de montrer que les différents produits naturels modulent systématiquement plusieurs gènes au cours de l'inhibition de la production d’AFB1. Notre étude montre que les produits naturels représentent une alternative possible pour limiter la contamination des aliments par l’AFB1. L'élucidation du mécanisme d'action des produits naturels ouvre de nouvelles perspectives sur les méthodes à employer pour inhiber la production de la toxine.Mycotoxin’s contamination represents an important source of food spoilage that has to be taken in consideration. For years pesticides, have been used as a common strategy to combat mycotoxin contamination. However, such products were also demonstrated to be harmful to humans and animals’ health. Therefore, it is necessary to find new strategies in order to avoid mycotoxin contamination and the use of natural products could be a promising alternative. Indeed, these compounds may be eco-friendly and several of them are demonstrated aseffective agents against toxin production. Nevertheless, their precise mechanism of action is poorly documented. One of the principal aim of this work consisted in the identification of natural sources capable to inhibit mycotoxin production and in the elucidation of their molecular mechanism of action. For that, we developed a molecular tool aiming the analysis of the impact of natural extracts on the expression of several genes related to Aflatoxin B1 synthesis in Aspergillus flavus. The study of this mycotoxin is an important issue since it is one of the most dangerous compounds inducing cancer in humans and animals. Taking advantage of the well-studied genome of Aspergillus flavus and considering that AFB1 production involves a great number of genetic elements, a q-PCR approach including 60 of the principal genes involved in toxin biosynthesis was developed. This tool simultaneously studies the entire AFB1 gene cluster but also 33 regulatory factors coding for external stimuli to which fungus is exposed. Using this molecular approach, the study of already known but also, new sources of anti-aflatoxigenic compounds was performed. The molecular mechanism of action of 2 isolated molecules and 3 whole plant extracts were determined and the impact of these compounds on the genes involved in AFB1 production was analysed. One of the innovative findings consisted in the demonstration that natural products systematically modulate several genes within AFB1’s inhibition. Taken together, our approach demonstrated that the use of natural products against mycotoxin production can represent an alternative strategy to inhibit food contamination. The elucidation of the mechanism of action of natural products allowed a better understanding of the fungal machinery through which toxin can be inhibited

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response

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 catalase enzymatic activity. In conclusion, these data demonstrated that piperine inhibits AFB1 production while positively modulating fungal antioxidant status in A. flavus

Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus

International audienceAflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark

Identification of the anti-aflatoxinogenic activity of Micromeria graeca and elucidation of its molecular mechanism in Aspergillus flavus

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