Recently, deoxynivalenol-3-sulfate (DON-3-sulfate) was proposed as a major DON metabolite in poultry. In the present work, the first LC-MS/MS based method for determination of DON-3-sulfate, deepoxy-DON-3-sulfate (DOM-3-sulfate), DON, DOM, DON sulfonates 1, 2, 3, and DOM sulfonate 2 in excreta samples of chickens and turkeys was developed and validated. To this end, DOM-3-sulfate was chemically synthesized and characterized by NMR and LC-HR-MS/MS measurements. Application of the method to excreta and chyme samples of four feeding trials with turkeys, chickens, pullets, and roosters confirmed DON-3-sulfate as the major DON metabolite in all poultry species studied. Analogously to DON-3-sulfate, DOM-3-sulfate was formed after oral administration of DOM both in turkeys and in chickens. In addition, pullets and roosters metabolized DON into DOM-3-sulfate. In vitro transcription/translation assays revealed DOM-3-sulfate to be 2000 times less toxic on the ribosome than DON. Biological recoveries of DON and DOM orally administered to broiler chickens, turkeys, and pullets were 74%–106% (chickens), 51%–72% (roosters), and 131%–151% (pullets). In pullets, DON-3-sulfate concentrations increased from jejunum chyme samples to excreta samples by a factor of 60. This result, put into context with earlier studies, indicates fast and efficient absorption of DON between crop and jejunum, conversion to DON-3-sulfate in intestinal mucosa, liver, and possibly kidney, and rapid elimination into excreta via bile and urine.

Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4. In the present study we show by in vitro transport studies that Rim2 mediates the transport of iron and other divalent metal ions across the mitochondrial inner membrane in a pyrimidine nucleotide-dependent fashion. Mutations in the proposed substrate-binding site of Rim2 prevent both pyrimidine nucleotide and divalent ion transport. These results document that Rim2 catalyses the co-import of pyrimidine nucleotides and divalent metal ions including ferrous iron. The deletion of RIM2 alone has no significant effect on mitochondrial iron supply, Fe–S protein maturation and haem synthesis. However, RIM2 deletion in mrs3/4Δ cells aggravates their Fe–S protein maturation defect. We conclude that under normal physiological conditions Rim2 does not play a significant role in mitochondrial iron acquisition, yet, in the absence of the main iron transporters Mrs3 and Mrs4, this carrier can supply the mitochondrial matrix with iron in a pyrimidine-nucleotide-dependent fashion.

In filamentous fungi such as Fusarium graminearum, disruption of multiple genes of interest in the same strain (e.g. to test for redundant gene function) is a difficult task due to the limited availability of reliable selection markers. We have created a series of transformation vectors that allow antibiotic-based selection of transformants and subsequent negative selection for marker removal using thymidine kinase fusions combined with the Cre-loxP system. The fusion genes contain commonly used C-terminal drug resistance markers, either nptII (G418), nat1 (nourseothricin), or hph (hygromycin B). These resistance genes are fused to the sequence encoding HSVtk (Herpes simplex virus thymidine kinase). Despite the presence of the 1 kb HSVtk gene (about ~30% increase in total marker size), there is only a slight reduction in transformation efficiency on a molar basis. The fusion genes expressed under the Trichoderma pyruvate kinase promoter also confer antibiotic resistance in Escherichia coli, allowing straightforward construction of disruption plasmids. For removal of the loxP flanked resistance cassettes, protoplasts of transformants are directly treated with purified Cre recombinase protein. Loss of the HSVtk containing cassette is selected by restoration of resistance to 5-fluoro-2-deoxyuridine (FdU). As a proof of principle, we demonstrated the efficiency of the HSVtk-based marker removal in Fusarium by reversing the disruption phenotype of the gene responsible for production of the red pigment aurofusarin. We first disrupted the FgPKS12 gene via integration of the loxP-flanked HSVtk-nptII cassette into the promoter or the first intron, thereby generating transformants with a white mycelium phenotype. Using Cre recombinase and FdU, the selection marker was subsequently removed, and the resulting transformants regained red pigmentation despite the remaining loxP site. We also found that it is possible to remove several unselected loxP-flanked cassettes with a single Cre protein treatment, as long as one of them contains a negative selectable HSVtk cassette. The negative selection system can also be used to introduce allele swaps into strains without leaving marker sequences, by first disrupting the gene of interest and then complementing the deletion in situ with genomic DNA containing a different allele.

A new type A trichothecene mycotoxin, NX-2, was previously reported to be produced by North American isolates of the cereal pathogen Fusarium graminearum. Here we describe the isolation and structural characterization of a rearrangement product, called NX2-M1, and related compounds with different acetylation patterns (NX3-M1 and NX4-M1). In the NX-M1 derivatives, the epoxide ring is opened, and a covalent bridge between C-10 and C-12 of the trichothecene backbone is formed. In vitro translation assays showed that NX3-M1 is less toxic for eukaryotic ribosomes than NX-3. NX3-M1 also has a greatly reduced cytotoxic potential on two tested human colon cell lines. Formation of NX3-M1 can therefore be regarded as a detoxification reaction. The related F. graminearum mycotoxin deoxynivalenol (DON), which is frequently occurring worldwide, is very stable during food processing. Testing NX-3 at different pH-values and temperature conditions, as well as under conditions that simulate the storage of infected grains and bread-making process, revealed a strongly reduced stability of NX-3 and concurrent formation of NX3-M1. Although the NX-3 formed in planta is as toxic as DON, the extensive formation of the non-toxic rearrangement product should be taken into account for risk assessment of this emerging food contaminant.

Increasing frequencies of 3-acetyl-deoxynivalenol (3-ADON)-producing strains of Fusarium graminearum (3-ADON chemotype) have been reported in North America and Asia. 3-ADON is nearly nontoxic at the level of the ribosomal target and has to be deacetylated to cause inhibition of protein biosynthesis. Plant cells can efficiently remove the acetyl groups of 3-ADON, but the underlying genes are yet unknown. We therefore performed a study of the family of candidate carboxylesterases (CXE) genes of the monocot model plant Brachypodium distachyon. We report the identification and characterization of the first plant enzymes responsible for deacetylation of trichothecene toxins. The product of the BdCXE29 gene efficiently deacetylates T-2 toxin to HT-2 toxin, NX-2 to NX-3, both 3-ADON and 15-acetyl-deoxynivalenol (15-ADON) into deoxynivalenol and, to a lesser degree, also fusarenon X into nivalenol. The BdCXE52 esterase showed lower activity than BdCXE29 when expressed in yeast and accepts 3-ADON, NX-2, 15-ADON and, to a limited extent, fusarenon X as substrates. Expression of these Brachypodium genes in yeast increases the toxicity of 3-ADON, suggesting that highly similar genes existing in crop plants may act as susceptibility factors in Fusarium head blight disease.

The Fusarium metabolite culmorin (1) is receiving increased attention as an “emerging mycotoxin”. It co-occurs with trichothecene mycotoxins and potentially influences their toxicity. Its ecological role and fate in plants is unknown. We synthesized sulfated and glucosylated culmorin conjugates as potential metabolites, which are expected to be formed in planta, and used them as reference compounds. An efficient procedure for the synthesis of culmorin sulfates was developed. Diastereo- and regioselective glucosylation of culmorin (1) was achieved by exploiting or preventing unexpected acyl transfer when using different glucosyl donors. The treatment of a wheat suspension culture with culmorin (1) revealed an in planta conversion of culmorin into culmorin-8-glucoside (6) and culmorin acetate, but no sulfates or culmorin-11-glucoside (7) was found. The treatment of wheat cells with the fungal metabolite 11-acetylculmorin (2) revealed its rapid deacetylation, but also showed the formation of 11-acetylculmorin-8-glucoside (8). These results show that plants are capable of extensively metabolizing culmorin.