Defects of the mitochondrial K

Fumonisins (FB) are among the most frequently detected mycotoxins in feedstuffs and finished feed, and recent data suggest that the functions of the gastrointestinal tract (GIT) in poultry species might be compromised at doses ranging from 10 to 20 mg/kg, close to field incidences and below the US and EU guidelines. Strategies are therefore necessary to reduce the exposure of poultry to FB. In the present study, we assessed the efficacy of fumonisin esterase FumD (EC 3.1.1.87, commercial name FUMzyme®) to cleave the tricarballylic acid side chains of FB, leading to the formation of non-toxic hydrolyzed fumonisins in the GIT of broiler chickens. Broiler chickens were fed for 14 d (7 to 21 d of age) 3 different diets (6 birds/cage, 6 cages/diet), i) control feed (negative control group), ii) feed contaminated with 10 mg FB/kg (FB group), and iii) feed contaminated with 10 mg FB/kg and supplemented with 100 units of FUMzyme®/kg (FB+FUMzyme® group). To determine the degree of reduction of FB in the GIT, 2 characteristics were analyzed. First, the sphinganine-to-sphingosine ratio in the serum and liver was determined as a biomarker of effect for exposure to FB. Second, the concentration of fumonisin B1 and its hydrolyzed forms was evaluated in the gizzard, the proximal and distal parts of the small intestine, and the excreta. Significantly reduced sphinganine-to-sphingosine ratios in the serum and liver of the FB+FUMzyme® group (serum: 0.15 ± 0.01; liver: 0.17 ± 0.01) compared to the FB group (serum: 0.20 ± 0.01; liver: 0.29 ± 0.03) proved that supplementation of broiler feed with FUMzyme® was effective in partially counteracting the toxic effect of dietary FB. Likewise, FB concentrations in digesta and excreta were significantly reduced in the FB+FUMzyme® group compared to the FB group (P < 0.05; up to 75%). FUMzyme® furthermore partially counteracted FB-induced up-regulation of cytokine gene expression (IL-8 and IL-10) in the jejunum. The FB group showed significantly higher gene expression of IL-8 and IL-10 compared to the negative control group (IL-8: fold change = 2.9 ± 1.1, P < 0.05; IL-10: fold change = 3.6 ± 1.4, P < 0.05), whereas IL-8 and IL-10 mRNA levels were not significantly different in the FB+FUMzyme®® group compared to the other 2 groups. In conclusion, FUMzyme® is suitable to detoxify FB in chickens and maintain gut functions.

Zearalenone (ZEN)-degrading enzymes are a promising strategy to counteract the negative effects of this mycotoxin in livestock. The reaction products of such enzymes need to be thoroughly characterized before technological application as a feed additive can be envisaged. Here, we evaluated the estrogenic activity of the metabolites hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed by hydrolysis of ZEN by the zearalenone-lactonase Zhd101p. ZEN, HZEN, and DHZEN were tested in two in vitro models, the MCF-7 cell proliferation assay (0.01–500 nM) and an estrogen-sensitive yeast bioassay (1–10,000 nM). In addition, we compared the impact of dietary ZEN (4.58 mg/kg) and equimolar dietary concentrations of HZEN and DHZEN on reproductive tract morphology as well as uterine mRNA and microRNA expression in female piglets (n = 6, four weeks exposure). While ZEN increased cell proliferation and reporter gene transcription, neither HZEN nor DHZEN elicited an estrogenic response, suggesting that these metabolites are at least 50–10,000 times less estrogenic than ZEN in vitro. In piglets, HZEN and DHZEN did not increase vulva size or uterus weight. Moreover, RNA transcripts altered upon ZEN treatment (EBAG9, miR-135a-5p, miR-187-3p and miR-204-5p) were unaffected by HZEN and DHZEN. Our study shows that both metabolites exhibit markedly reduced estrogenicity in vitro and in vivo, and thus provides an important basis for further evaluation of ZEN-degrading enzymes.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and β-zearalenol (β-ZEL) was detected in lower concentrations. ZEN, α-ZEL and β-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and β-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.

Fumonisins are among the most prevalent mycotoxins in feedstuffs. They disrupt the sphingolipid metabolism, thereby inducing a plethora of toxic effects in livestock. Supplementation with mycotoxin-degrading enzymes is a promising strategy for the detoxification of feedstuffs in the animals’ gastrointestinal tract. Here, we evaluated the suitability of the fumonisin esterase FumD as a feed additive (FUMzyme®) for the prevention of fumonisin toxicity in pigs by using a combination of different fumonisin biomarkers (sphinganine to sphingosine (Sa/So) ratio in serum and organs, concentrations of fumonisin B1 and hydrolysed derivatives in urine and faeces). In a pre-trial, we exposed pigs to 30 mg/kg fumonisins in feed and found the minimum effective dose of FUMzyme to be 15 U/kg. In a second trial we investigated the long-term efficacy of this minimum effective FUMzyme dose to counteract toxic effects elicited by 6 weeks of exposure to 2.5 mg/kg fumonisins in a diet containing naturally contaminated maize. Supplementation of feed with the minimum effective FUMzyme dose prevented an increase in the Sa/So ratio in serum and kidneys of fumonisin exposed pigs. The Sa/So ratio in serum proved to be the most reliable biomarker. The fumonisin pattern in faeces was less suitable as biomarker for assessing the efficacy of FUMzyme due to natural gastrointestinal hydrolysis of fumonisins. Analysis of urine samples provided additional information about gastrointestinal fumonisin hydrolysis before fumonisin absorption, but was analytically challenging because of low urinary fumonisin concentrations.

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products); (b) enzymatic detoxification of zearalenone (ZEN; one product, ZenA); and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite α-zearalenol (α-ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes.

Under conditions of environmental stress, the plasma membrane is involved in several regulatory processes to promote cell survival, like maintenance of signaling pathways, cell wall organization and intracellular ion homeostasis. PUN1 encodes a plasma membrane protein localizing to the ergosterol-rich membrane compartment occupied also by the arginine permease Can1. We found that the PUN1 (YLR414c) gene is transcriptionally induced upon metal ion stress. Northern blot analysis of the transcriptional regulation of PUN1 showed that the calcium dependent transcription factor Crz1p is required for PUN1 induction upon heavy metal stress. Here we report that mutants deleted for PUN1 exhibit increased metal ion sensitivity and morphological abnormalities. Microscopical and ultrastructural observations revealed a severe cell wall defect of pun1∆ mutants. By using chemical cross-linking, Blue native electrophoresis, and co-immunoprecipitation we found that Pun1p forms homo-oligomeric protein complexes. We propose that Pun1p is a stress-regulated factor required for cell wall integrity, thereby expanding the functional significance of lateral plasma membrane compartments.