Bases moléculaires de la voie de biosynthèse de la patuline, mycotoxine produite par Byssochlamys nivea et Penicillium griseofulvum

La patuline constitue un contaminant chimique toxique fréquemment rencontré dans les produits issus de la transformation des fruits, notamment des pommes. Cette toxine essentiellement produite par Penicillium expansum et Byssochlamys nivea fait l'objet d'une réglementation européenne récente (N°1425/2003). Contrairement à certaines mycotoxines règlementées telles que les aflatoxines, les trichothécènes ou les fumonisines, la génétique de la voie de biosynthèse de la patuline est fort mal connue, bien que cette voie ait été relativement bien caractérisée du point de vue chimique. Deux espèces toxinogènes, Byssochlamys nivea et Penicillium griseofulvum ont été étudiées en tant qu'espèces modèles. Trois gènes impliqués dans la synthèse de la patuline ont été isolés de B. nivea, et entièrement séquencés lors de ce travail. Le premier gène 6msas isolé code pour une polycétide synthase, l'acide 6-methylsalicylique synthase, intervenant au début de la cascade enzymatique conduisant à la synthèse de la patuline. Le deuxième gène idh coderait pour une alcool déshydrogénase impliquée dans la transformation de l'isoépoxydon en phyllostine, deux autres précurseurs de la patuline. En amont de ce dernier gène, sur le brin complémentaire, un gène abc codant pour un transporteur actif de la famille des ABC transporteurs a été localisé, isolé et entièrement séquencé chez Penicillium griseofulvum, puis chez B. nivea et P. expansum. La présence d'un tel gène ne semble pas aberrante puisqu'il a été montré que certains transporteurs actifs faisaient partie de l'arsenal de résistance développé par les champignons pour ne pas subir les effets délétères des toxines qu'ils synthétisent. D'après les données brutes du séquençage du génome d'Aspergillus clavatus, autre espèce de patuline, le complexe génique abc/idh serait distant de 10 kb du gène 6msas. L'analyse de la région avoisinant ces gènes dévoile l'existence chez A. clavatus d'un cluster de gènes constitué d'au moins 12 gènes en comptant les trois gènes préalablement identifiés, potentiellement impliqués dans la synthèse de la mycotoxine. Parmi ces gènes, plusieurs codent pour des enzymes dont l'implication semble évidente au regard des données actuellement disponibles sur la caractérisation chimique de la voie de biosynthèse de la patuline. Du point de vue application, ces travaux ont d'ores et déjà apportés des réponses concrètes à des problèmes industriels. Une étude réalisée sur un panel relativement divers de souches d'origine géographique différente de Byssochlamys nivea et Byssochlamys fulva, conclut à la non-production de patuline par B. fulva sur des bases analytiques et génétiques. Cette absence de production est due à l'absence d'au moins deux gènes, 6msas et idh. B fulva fréquemment isolé des fruits ne représente donc pas une source de contamination des pommes par la patuline dans les filières de transformation. Penicillium expansum et Byssochlamys nivea sont donc considérés comme les principales sources de contamination des pommes par la patuline. ABSTRACT : Patulin is toxic chemical contaminant produced by several species of mould (Penicillium griseofulvum, P.expansum, Byssochlamys nivea, Aspergillus clavatus…). Exposure to this mycotoxin is associated with immunological, neurological and gastrointestinal outcomes. Assessment of the health risks due to patulin consumption by humans lead many countries to regulate its amounts in food. In Europe, a maximum level has been established of 50 μg per kg for apple juice, cider and a maximum level of 10 μg/kg for all dietary products intended for infants and young children. Unlike other regulated mycotoxins (aflatoxins, trichothecens and fumonisines), the knowledge regarding the patulin biosynthesis is so far limited to the chemical characterization of patulin precursors and to the identification of two relevant genes from Penicillium griseofulvum (6- methylsalicylic acid synthase (6msas) and isoepoxydon dehydrogenase (idh). Two toxinogenic species Byssochlamys nivea and Penicillium griseofulvum have been studied in this work. Three genes (6msas, idh and abc) that belong to the patulin biosynthetic pathway were isolated, and wholly sequenced in B. nivea. These genes are coding respectively for 6- methylsalicylic acid synthase, the first enzyme involved in patulin biosynthesis, isoepoxydon dehydrogenase which allowed the isoepoxydon transformation in phyllostin and an ABC (ATP Binding Cassette) transporter. This latest gene is located on the anti-sense strand, upstream of the idh gene, and it has been isolated also from Penicillium griseofulvum and Penicillium expansum. This transporter could be responsible for the active efflux of endogenously produced patulin and contribute to self protection against patulin in producing fungi. After comparison with data from the Aspergillus clavatus genome sequencing program performed by TIGR (The Institute for Genomic Research), we noticed that the abc/idh genes complex and 6msas gene are 10 kb away from each other. We performed a bioinformatic analysis of the regions located upstream and downstream of this 10 kb size fragment, and established the presence of 9 additional genes. Their potential involvement in the synthesis of the toxin has been discussed. Finally, this fundamental work could answer to industrial problems. A study performed on 19 different strains of B. nivea and B. fulva showed that Byssochlamys fulva don't produce patulin and that its inability to do so could be explained by the lack of both 6msas and idh genes. In conclusion, B. fulva clearly lacks several biochemical potencies to be responsible for the occurrence of patulin in fruit

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae

Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298 bp polyketide synthase gene ‘‘aomsas” has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40–56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant ‘‘aoDmsas” of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone,but also 6-methylsalicylic acid. The genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aoDmsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone

Characterization of a cytochrome P450 monooxygenase gene involved in the biosynthesis of geosmin in Penicillium expansum

Geosmin is a terpenoid, an earthy-smelling substance associated with off-flavors in water and wine. The biosynthesis of geosmin is well characterized in bacteria, but little is known about its production in eukaryotes, especially in filamentous fungi. The origin of geosmin in grapevine is largely attributable to the presence of Penicillium expansum on grapes. Herein, we describe the characterization of “gpe1”, a gene encoding a cytochrome P450 monooxygenase probably involved in the biosynthesis of geosmin in this species. A gpe1knockout mutant of P. expansum M2230 lost the capacity to produce geosmin, while the genetically complemented mutant restored it. The deduced gpe1 protein sequence shows identities with other cytochrome P450 monooxygenases involved in diterpene biosynthesis. These enzymes catalyze the addition of hydroxyl groups to the diterpene compounds. gpe1protein could work in the same way, with sesquiterpenes as substrates. This gene seems to be only present in geosmin-producing Penicillium species. To our knowledge, this is the first characterization of a fungal gene encoding an enzyme involved in geosmin biosynthesis

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

Toxicology of mycotoxins, hazards and risks in human and animal food

Mycotoxins are secondary metabolites produced on plants either in the field or during storage. These toxins are found as natural contaminants on numerous foods and feeds of plant origin, such as cereals, fruits, nuts, almonds, grains, fodder, as well as processed foods and feeds using these ingredients. The toxicity of mycotoxins varies, ranging from hepatotoxic or even carcinogenic (aflatoxins) effects, to estrogenic (zearalenone), immunotoxic (patulin, trichothecenes, fumonisins), nephrotoxic (ochratoxin A) and neurotoxic (tremorgens) effects. Their toxicity can also be caused by the presence of mycotoxin residues in products deriving from animals fed with contaminated feedstuffs. The mycotoxic risk is difficult to evaluate, as mycotoxin are natural contaminants impossible to eliminate, fungal contaminations are difficult to control, and one mould may produce several toxins. Consequently, further research is needed to improve current knowledge on the toxicity of these products, particularly when various mycotoxins are combined, either together or with other toxins or pathogens.Les mycotoxines sont des produits du métabolisme secondaire de moisissures pouvant se développer sur la plante au champ ou en cours de stockage. Ces toxines se retrouvent à l'état de contaminants naturels de nombreuses denrées d'origine végétale : céréales, fruits, noix, amandes, grains, fourrages ainsi que d'aliments composés et manufacturés issus de ces filières. La toxicité des mycotoxines se révèle lors des mycotoxicoses des animaux d'élevage. Elle est variable, certaines exerçant un pouvoir hépa-totoxique voire cancérogène (aflatoxines), d'autres se révélant oestrogèniques (zéaralénone), immunotoxiques (patuline, trichothécènes, fumonisines), néphrotoxiques (ochratoxine A) ou neurotoxiques (trémorgènes). Un autre aspect de leur toxicité est la prise en compte des résidus présents dans les productions issues d'animaux ayant consommé une alimentation contaminée. L'évaluation du risque mycotoxique demeure délicate car ce risque est d'essence naturelle, l'homme n'en maîtrisant pas la survenue ; il est pernicieux car la contamination fongique est difficilement contrôlable et enfin il peut être multiple en raison de la possible association d'effets de toxines produites par une même moisissure. Devant ce constat, il convient de poursuivre une activité de recherche soutenue afin d'améliorer encore nos connaissances sur la toxicité de ces dérivés et notamment dans les cas d'associations entre mycotoxines ou entre toxines et agents pathogènes infectieux

Homeobox transcription factor HbxA influences expression of over one thousand genes in the model fungus \u3ci\u3eAspergillus nidulans\u3c/i\u3e

In fungi, conserved homeobox-domain proteins are transcriptional regulators governing development. In Aspergillus species, several homeobox-domain transcription factor genes have been identified, among them, hbxA/hbx1. For instance, in the opportunistic human pathogen Aspergillus fumigatus, hbxA is involved in conidial production and germination, as well as virulence and secondary metabolism, including production of fumigaclavines, fumiquinazolines, and chaetominine. In the agriculturally important fungus Aspergillus flavus, disruption of hbx1 results in fluffy aconidial colonies unable to produce sclerotia. hbx1 also regulates production of aflatoxins, cyclopiazonic acid and aflatrem. Furthermore, transcriptome studies revealed that hbx1 has a broad effect on the A. flavus genome, including numerous genes involved in secondary metabolism. These studies underline the importance of the HbxA/Hbx1 regulator, not only in developmental processes but also in the biosynthesis of a broad number of fungal natural products, including potential medical drugs and mycotoxins. To gain further insight into the regulatory scope of HbxA in Aspergilli, we studied its role in the model fungus Aspergillus nidulans. Our present study of the A. nidulans hbxA-dependent transcriptome revealed that more than one thousand genes are differentially expressed when this regulator was not transcribed at wild-type levels, among them numerous transcription factors, including those involved in development as well as in secondary metabolism regulation. Furthermore, our metabolomics analyses revealed that production of several secondary metabolites, some of them associated with A. nidulans hbxA-dependent gene clusters, was also altered in deletion and overexpression hbxA strains compared to the wild type, including synthesis of nidulanins A, B and D, versicolorin A, sterigmatocystin, austinol, dehydroaustinol, and three unknown novel compounds

Homeobox transcription factor HbxA influences expression of over one thousand genes in the model fungus \u3ci\u3eAspergillus nidulans\u3c/i\u3e

In fungi, conserved homeobox-domain proteins are transcriptional regulators governing development. In Aspergillus species, several homeobox-domain transcription factor genes have been identified, among them, hbxA/hbx1. For instance, in the opportunistic human pathogen Aspergillus fumigatus, hbxA is involved in conidial production and germination, as well as virulence and secondary metabolism, including production of fumigaclavines, fumiquinazolines, and chaetominine. In the agriculturally important fungus Aspergillus flavus, disruption of hbx1 results in fluffy aconidial colonies unable to produce sclerotia. hbx1 also regulates production of aflatoxins, cyclopiazonic acid and aflatrem. Furthermore, transcriptome studies revealed that hbx1 has a broad effect on the A. flavus genome, including numerous genes involved in secondary metabolism. These studies underline the importance of the HbxA/Hbx1 regulator, not only in developmental processes but also in the biosynthesis of a broad number of fungal natural products, including potential medical drugs and mycotoxins. To gain further insight into the regulatory scope of HbxA in Aspergilli, we studied its role in the model fungus Aspergillus nidulans. Our present study of the A. nidulans hbxA-dependent transcriptome revealed that more than one thousand genes are differentially expressed when this regulator was not transcribed at wild-type levels, among them numerous transcription factors, including those involved in development as well as in secondary metabolism regulation. Furthermore, our metabolomics analyses revealed that production of several secondary metabolites, some of them associated with A. nidulans hbxA-dependent gene clusters, was also altered in deletion and overexpression hbxA strains compared to the wild type, including synthesis of nidulanins A, B and D, versicolorin A, sterigmatocystin, austinol, dehydroaustinol, and three unknown novel compounds

Verruculogen associated with Aspergillus fumigatus hyphae and conidia modifies the electrophysiological properties of human nasal epithelial cells

BACKGROUND: The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds. RESULTS: We fractionated the organic phase of filtrate from 3-day old A. fumigatus cultures using high-performance liquid chromatography. The different fractions were tested for their ability to modify the electrophysiological properties of HNEC in an in vitro primary culture model. The fraction collected between 20 and 30 min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. Only verruculogen (10(-8 )M) had effects similar to the whole filtrate. We verified that verruculogen was produced by a collection of 67 human, animal, plant and environmental A. fumigatus isolates. Using MS-MS analysis, we found that verruculogen was associated with both mycelium and conidia extracts. CONCLUSION: Verruculogen is a secondary metabolite that modifies the electrophysiological properties of HNEC. The role of these modifications in the colonization and invasion of the respiratory epithelium by A. fumigatus on first contact with the epithelium remains to be determined

Verruculogen associated with Aspergillus fumigatus hyphae and conidia modifies the electrophysiological properties of human nasal epithelial cells

BACKGROUND: The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds. RESULTS: We fractionated the organic phase of filtrate from 3-day old A. fumigatus cultures using high-performance liquid chromatography. The different fractions were tested for their ability to modify the electrophysiological properties of HNEC in an in vitro primary culture model. The fraction collected between 20 and 30 min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. Only verruculogen (10(-8 )M) had effects similar to the whole filtrate. We verified that verruculogen was produced by a collection of 67 human, animal, plant and environmental A. fumigatus isolates. Using MS-MS analysis, we found that verruculogen was associated with both mycelium and conidia extracts. CONCLUSION: Verruculogen is a secondary metabolite that modifies the electrophysiological properties of HNEC. The role of these modifications in the colonization and invasion of the respiratory epithelium by A. fumigatus on first contact with the epithelium remains to be determined