Combination of Isotope Labeling and Molecular Networking of Tandem Mass Spectrometry Data To Reveal 69 Unknown Metabolites Produced by Penicillium nordicum.

The 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.

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.

Aspergillus korhogoensis, a Novel Aflatoxin Producing Species from the Côte d'Ivoire.

Several strains of a new aflatoxigenic species of Aspergillus, A. korhogoensis, were isolated in the course of a screening study involving species from section Flavi found contaminating peanuts (Arachis hypogaea) and peanut paste in the Côte d'Ivoire. Based on examination of four isolates, this new species is described using a polyphasic approach. A concatenated alignment comprised of nine genes (ITS, benA, cmdA, mcm7, amdS, rpb1, preB, ppgA, and preA) was subjected to phylogenetic analysis, and resulted in all four strains being inferred as a distinct clade. Characterization of mating type for each strain revealed A. korhogoensis as a heterothallic species, since three isolates exhibited a singular MAT1-1 locus and one isolate exhibited a singular MAT1-2 locus. Morphological and physiological characterizations were also performed based on their growth on various types of media. Their respective extrolite profiles were characterized using LC/HRMS, and showed that this new species is capable of producing B- and G-aflatoxins, aspergillic acid, cyclopiazonic acid, aflavarins, and asparasones, as well as other metabolites. Altogether, our results confirm the monophyly of A. korhogoensis, and strengthen its position in the A. flavus clade, as the sister taxon of A. parvisclerotigenus.

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 natural ecosystems 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 wheat grains labeled with stable isotopes: (i) natural grains (99% 12C); (ii) grains enriched with 97% of 13C; and (iii) grains enriched with 53% of 13C and 97% of 15N. Analyses performed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) enabled the specific detection of fungal metabolites, and the unambiguous characterization of their chemical formulas. In this way, 98 secondary metabolites were detected and their chemical formulas were determined. Of these, only 18 identifications could be made based on databases, the literature and mass spectrometry fragmentation experiments, with the result that 80 were totally unknown. Molecular networks were generated to analyze these results, leading to the characterization by MSn experiments of a new fungisporin produced by P. verrucosum. More generally, this article provides precise mass spectrometric data about all these compounds for further studies of the Penicillium metabolome.

The gene PatG involved in the biosynthesis pathway of patulin, a food-borne mycotoxin, encodes a 6-methylsalicylic acid decarboxylase.

Patulin is a mycotoxin produced by fungal genera such as Aspergillus, Penicillium and Byssochlamys. It induces neurological, gastrointestinal and immunological effects, which is why patulin belongs to a short list of mycotoxins whose level in food is regulated in many countries around the world. Recently, a cluster gathering 15 genes involved in the biosynthesis of patulin has been identified in Aspergillus clavatus, but so far, only 4 genes encoding 6-methylsalicylic acid synthase, m-cresol hydroxylase, m-hydroxybenzyl alcohol hydroxylase and isoepoxydon dehydrogenase have been characterized. Previous studies have shown the involvement of a decarboxylase in the transformation of 6-methylsalicylic acid, the first stable patulin precursor, into m-cresol. In this study a putative decarboxylase gene, PatG, was identified in the genome sequence of A. clavatus. This gene is located near two P450 cytochrome genes PatH and PatI responsible respectively for the hydroxylation of m-cresol and m-hydroxybenzyl alcohol. This decarboxylase encoded by PatG (ACLA_093620) consists of 325 amino acids. The search for putative conserved domain revealed that the gene product belongs to the AminoCarboxyMuconate Semialdehyde Decarboxylase (ACMSD) related protein family. This family includes decarboxylases such as the γ-resorcylate decarboxylase or o-pyrocatechuate decarboxylase. The substrates of these enzymes display strong structural similarities with 6-methylsalicylic acid. PatG was strongly expressed during patulin production whereas it was very weakly expressed in non-patulin permissive conditions. The coding sequence was used to enable heterologous expression of functional enzymes in Saccharomyces cerevisiae. The presence of decarboxylase was confirmed by Western blot. The bioconversion assays showed that PATG catalyzed the decarboxylation of 6-methylsalicylic acid into m-cresol. These results confirm for the first time that 6-methylsalicylic acid is the substrate for PATG, the 6-methylsalicylic acid decarboxylase. With this study, the four genes involved in the four first steps of patulin biosynthesis pathway (acetate→gentisyl alcohol) are now identified.

New untargeted metabolic profiling combining mass spectrometry and isotopic labeling: application on Aspergillus fumigatus grown on wheat.

Characterization 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% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. 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 (MS(n)) 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.

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.

Molecular cloning and functional characterization of two CYP619 cytochrome P450s involved in biosynthesis of patulin in Aspergillus clavatus.

Patulin is an acetate-derived tetraketide mycotoxin produced by several fungal species, especially Aspergillus, Penicillium and Byssochlamys species. The health risks due to patulin consumption by humans have led many countries to regulate it in human food. Previous studies have shown the involvement of cytochrome P450 monooxygenases in the hydroxylation of two precursors of patulin, m-cresol and m-hydroxybenzylalcohol. In the present study, two cytochrome P450 genes were identified in the genome sequence of Aspergillus clavatus, a patulin-producing species. Both mRNAs were strongly co-expressed during patulin production. CYP619C2, encoded by the first gene, consists of 529 aa, while the second cytochrome, CYP619C3, consists of 524 aa. The coding sequences were used to perform the heterologous expression of functional enzymes in Saccharomyces cerevisiae. The bioconversion assays showed that CYP619C3 catalysed the hydroxylation of m-cresol to yield m-hydroxybenzyl alcohol. CYP619C2 catalysed the hydroxylation of m-hydroxybenzyl alcohol and m-cresol to gentisyl alcohol and 2,5-dihydroxytoluene (toluquinol), respectively. Except for the last compound, all enzyme products are known precursors of patulin. Taken together, these data strongly suggest the involvement of CYP619C2 and CYP619C3 in the biosynthesis of patulin. CYP619C2 and CYP619C3 are located near to two other genes involved in patulin biosynthesis, namely the 6-methylsalicylic acid synthase (6msas) and isoepoxydon dehydrogenase (idh) genes. The current data associated with an analysis of the sequence of A. clavatus suggest the presence of a cluster of 15 genes involved in patulin biosynthesis.

The inability of Byssochlamys fulva to produce patulin is related to absence of 6-methylsalicylic acid synthase and isoepoxydon dehydrogenase genes.

Byssochlamys species are responsible for spoilage and degradation of fruits and silages. Under specific conditions they are able to produce mycotoxins. The aim of this study was to evaluate the potential of 19 different strains of Byssochlamys nivea and Byssochlamys fulva to produce patulin in relation with the presence of two genes involved in the patulin biosynthesis pathways in the genome of these fungal strains. The strains were characterized by macroscopic, microscopic examinations, internal transcribed spacer (ITS) rRNA and beta-tubulin fragment amplification and sequencing. All of the 8 B. nivea strains tested produced patulin. By contrast, none of the 11 strains of B. fulva produce this toxin. Two genes of the patulin biosynthetic pathway, a polyketide synthase (pks) and the isoepoxydon dehydrogenase (idh) were cloned from B. nivea. The deduced amino acid sequence of the polyketide synthase was 74% identical to the 6-methylsalicylic acid synthase gene of Penicillium griseofulvum and had the five functional domains characteristic of fungal type I polyketide synthases (beta-ketosynthase, acyltransferase, dehydratase, beta-ketoreductase and acyl carrier protein). The complete coding sequence of idh gene displayed after translation 88% of identity with P. griseofulvum IDH and 85% with P. expansum IDH, respectively. Both pks and idh messengers were strongly co-expressed during the production of 6-methylsalicylic acid and patulin. The presence of these genes was then investigated in the genome of B. nivea and B. fulva strains by PCR. All B. nivea strains possess the two genes, by contrast none of the B. fulva strains display these genes. The absence of 6-methylsalicylic acid and isoepoxydon dehydrogenase genes can explain the inability of B. fulva to produce patulin. In conclusion, B. fulva don't seem to be responsible for the occurrence of patulin by lack of genes.

Byssochlamys nivea as a source of mycophenolic acid.

Byssochlamys 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.