Impact of veA on the development, aggressiveness, dissemination and secondary metabolism of Penicillium expansum

Penicillium expansum, the causal agent of blue mould disease, produces the mycotoxins patulin and citrinin amongst other secondary metabolites. Secondary metabolism is associated with fungal development, which responds to numerous biotic and abiotic external triggers. The global transcription factor VeA plays a key role in the coordination of secondary metabolism and differentiation processes in many fungal species. The specific role of VeA in P. expansum remains unknown. A null mutant PeΔveA strain and a complemented PeΔveA:veA strain were generated in P. expansum and their pathogenicity on apples was studied. Like the wild‐type and the complemented strains, the null mutant PeΔveA strain was still able to sporulate and to colonize apples, but at a lower rate. However, it could not form coremia either in vitro or in vivo, thus limiting its dissemination from natural substrates. The impact of veA on the expression of genes encoding proteins involved in the production of patulin, citrinin and other secondary metabolites was evaluated. The disruption of veA drastically reduced the production of patulin and citrinin on synthetic media, associated with a marked down‐regulation of all genes involved in the biosynthesis of the two mycotoxins. Moreover, the null mutant PeΔveA strain was unable to produce patulin on apples. The analysis of gene expression revealed a global impact on secondary metabolism, as 15 of 35 backbone genes showed differential regulation on two different media. These findings support the hypothesis that VeA contributes to the pathogenicity of P. expansum and modulates its secondary metabolism

Trypacidin, a Spore-Borne Toxin from Aspergillus fumigatus, Is Cytotoxic to Lung Cells

Inhalation of Aspergillus fumigatus conidia can cause severe aspergillosis in immunosuppressed people. A. fumigatus produces a large number of secondary metabolites, some of which are airborne by conidia and whose toxicity to the respiratory tract has not been investigated. We found that spores of A. fumigatus contain five main compounds, tryptoquivaline F, fumiquinazoline C, questin, monomethylsulochrin and trypacidin. Fractionation of culture extracts using RP-HPLC and LC-MS showed that samples containing questin, monomethylsulochrin and trypacidin were toxic to the human A549 lung cell line. These compounds were purified and their structure verified using NMR in order to compare their toxicity against A549 cells. Trypacidin was the most toxic, decreasing cell viability and triggering cell lysis, both effects occurring at an IC50 close to 7 µM. Trypacidin toxicity was also observed in the same concentration range on human bronchial epithelial cells. In the first hour of exposure, trypacidin initiates the intracellular formation of nitric oxide (NO) and hydrogen peroxide (H2O2). This oxidative stress triggers necrotic cell death in the following 24 h. The apoptosis pathway, moreover, was not involved in the cell death process as trypacidin did not induce apoptotic bodies or a decrease in mitochondrial membrane potential. This is the first time that the toxicity of trypacidin to lung cells has been reported

Byssochlamys nivea as a source of mycophenolic acid

International audienceByssochlamys species are responsible for spoilage and degradation of fruits and silages and can also produce the mycotoxin patulin. We analyzed secondary metabolite production by Byssochlamys nivea. Mycophenolic acid and its precursors, 5-methylorsellinic acid and 5,7-dihydroxy-4-methylphthalide, were identified in all of the B. nivea strains that we examined

New untargeted metabolic profiling combining mass spectrometry and isotopic labeling: Application on <em>Aspergillus fumigatus</em> grown on wheat

International audienceCharacterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus, an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% 12C), (2) grains enriched 96.8% with 13C, (3) grains enriched with 53.4% with 13C and 96.8% with 15N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography−high-resolution mass spectrometry (HPLC−HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MSn) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC− HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins

Combination of isotope labeling and molecular networking of tandem mass spectrometry data to reveal 69 unknown metabolites produced by Penicillium nordicum

International audienceThe secondary metabolome of Penicillium nordicum is poorly documented despite its frequent detection on contaminated food and its capacity to produce toxic metabolites such as ochratoxin A. To characterize metabolites produced by this fungi, we combined a full stable isotopes labeling with the dereplication of tandem mass spectrometry (MS/MS) data by molecular networking. First, the untargeted metabolomic analysis by high-resolution mass spectrometry of a double stable isotope labeling of P. nordicum enabled the specific detection of its metabolites and the unambiguous determination of their elemental composition. Analyses showed that infection of substrate by P. nordicum lead to the production of at least 92 metabolites and that 69 of them were completely unknown. Then, curated molecular networks of MS/MS data were generated with GNPS and MetGem, specifically on the features of interest, which allowed highlighting 13 fungisporin-related metabolites that had not previously been identified in this fungus and 8 that had never been observed in any fungus. The structures of the unknown compounds, namely, a native fungisporin and seven linear peptides, were characterized by tandem mass spectrometry experiments. The analysis of P. nordicum growing on its natural substrates, i.e. pork ham, turkey ham, and cheese, demonstrated that 10 of the known fungisporin-related metabolites and three of the new metabolites were also synthesized. Thus, the curation of data for molecular networking using a specific detection of metabolites of interest with stable isotopes labeling allowed the discovery of new metabolites produced by the food contaminant P. nordicum

Genotoxicity of aflatoxins and their precursors in human cells

International audienceAflatoxins are found as food contaminant and some of them demonstrate a carcinogenic effect. The aflatoxins biosynthetic pathway involves 15 successive steps. The aim of this study was to compare the toxicity of aflatoxins and their precursors in three human cell lines. We tested the four aflatoxins and two of their metabolites; three early metabolic precursors and two late biosynthetic precursors. Cyclopiazonic acid, synthesized in parallel with aflatoxins, was also tested. The cytotoxicity and the genotoxicity was evaluated with the γH2AX assay in three human cell lines with different bioactivation capacities. Our results indicated that the most genotoxic chemicals in the three cell lines were in decreasing order sterigmatocystin (ST), aflatoxin B1 (AFB1), aflatoxicol (AFL), aflatoxin G1 (AFG1) and versicolorin A (VERA). Aflatoxin M1 (AFM1) demonstrated genotoxic property in only one cell line. The other tested compounds did not demonstrate any genotoxic activity. Overall, our results suggested different genotoxic mechanisms of action for the tested compounds, involving specific bioactivation pathways. Moreover, some metabolic precursors of aflatoxins demonstrated genotoxic potential equivalent or greater to AFB1. This should be taking into account for the development of new strategies intended to reduce the aflatoxins exposure and for human risk assessment

Evidencing 98 secondary metabolites of Penicillium verrucosum using substrate isotopic labeling and high-resolution mass spectrometry

Industrial applications of fungal compounds, coupled with the emergence of fungal threats to naturalecosystems and public health, have increased interest in filamentous fungi. Among all pathogenic fungi,Penicillium verrucosum is one of the most common mold-infecting stored cereals in temperate regions.However, it is estimated that 80% of fungal secondary metabolites remain unknown. To detect new P.verrucosum compounds, an untargeted metabolomic approach was applied to fungus grown on wheatgrains labeled with stable isotopes: (i) natural grains (99%12C); (ii) grains enriched with 97% of13C; and(iii) grains enriched with 53% of13C and 97% of15N. Analyses performed by high-performance liquidchromatography coupled with high-resolution mass spectrometry (HPLC–HRMS) enabled the specificdetection of fungal metabolites, and the unambiguous characterization of their chemical formulas. Inthis way, 98 secondary metabolites were detected and their chemical formulas were determined. Ofthese, only 18 identifications could be made based on databases, the literature and mass spectrometryfragmentation experiments, with the result that 80 were totally unknown. Molecular networks weregenerated to analyze these results, leading to the characterization by MSnexperiments of a new fungis-porin produced by P. verrucosum. More generally, this article provides precise mass spectrometric dataabout all these compounds for further studies of the Penicillium metabolome

Analysis of the contrast between natural occurrence of toxigenic Aspergilli of the Flavi section and aflatoxin B1 in cassava

International audienceAflatoxin B1 (AFB1) is a carcinogenic mycotoxin produced by Aspergilli of the section Flavi that may contaminate food, in the field or during storage. Cassava represents an important staple food in sub-Saharan Africa. The analysis of aflatoxigenic fungi in 36 cassava samples obtained from producers in Benin indicated that 40% were contaminated by Aspergilli of the section Flavi. Upon morphological and molecular characterization of the 20 isolates, 16 belonged to Aspergillus flavus, 2 to Aspergillus parvisclerotigenus and 2 to Aspergillus novoparasiticus. This is the first time that this latter species is isolated from food. Although most of these isolates were toxigenic on synthetic media, no AFB1 contamination was observed in these cassava samples. In order to determine the action of cassava on AFB1 synthesis, a highly toxigenic strain of A. flavus, was inoculated onto fresh cassava and despite a rapid development, no AFB1 was produced. The anti-aflatoxin property was observed with cassava from different geographical origins and on other aflatoxigenic strains of the section Flavi, but it was lost after heating, sun drying and freezing. Our data suggest that fresh cassava is safe regarding AFB1 contamination, however, processing may alter its ability to block toxinogenesis leading to secondary contamination. (C) 2013 Elsevier Ltd. All rights reserved