Patulin Biodegradation Mechanism Study in Pichia guilliermondii S15-8 Based on PgSDR-A5D9S1.

Patulin contamination has become a bottleneck problem in the safe production of fruit products, although biodegradation technology shows potential application value in patulin control. In the present study, the patulin biodegradation mechanism in a probiotic yeast, Pichia guilliermondii S15-8, was investigated. Firstly, the short-chain dehydrogenase PgSDR encoded by gene A5D9S1 was identified as a patulin degradation enzyme, through RNA sequencing and verification by qRT-PCR. Subsequently, the exogenous expression system of the degradation protein PgSDR-A5D9S1 in E. coli was successfully constructed and demonstrated a more significant patulin tolerance and degradation ability. Furthermore, the structure of PgSDR-A5D9S1 and its active binding sites with patulin were predicted via molecular docking analysis. In addition, the heat-excited protein HSF1 was predicted as the transcription factor regulating the patulin degradation protein PgSDR-A5D9S1, which may provide clues for the further analysis of the molecular regulation mechanism of patulin degradation. This study provides a theoretical basis and technical support for the industrial application of biodegradable functional strains.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Immunosuppressive effects of the mycotoxin patulin in macrophages.

Patulin (PAT) is a fungi-derived secondary metabolite produced by numerous fungal species, especially within Aspergillus, Byssochlamys, and Penicillium genera, amongst which P. expansum is the foremost producer. Similar to other fungi-derived metabolites, PAT has been shown to have diverse biological features. Initially, PAT was used as an effective antimicrobial agent against Gram-negative and Gram-positive bacteria. Then, PAT has been shown to possess immunosuppressive properties encompassing humoral and cellular immune response, immune cell function and activation, phagocytosis, nitric oxide and reactive oxygen species production, cytokine release, and nuclear factor-κB and mitogen-activated protein kinases activation. Macrophages are a heterogeneous population of immune cells widely distributed throughout organs and connective tissue. The chief function of macrophages is to engulf and destroy foreign bodies through phagocytosis; this ability was fundamental to his discovery. However, macrophages play other well-established roles in immunity. Thus, considering the central role of macrophages in the immune response, we review the immunosuppressive effects of PAT in macrophages and provide the possible mechanisms of action.

Patulin induces ROS-dependent cardiac cell toxicity by inducing DNA damage and activating endoplasmic reticulum stress apoptotic pathway.

Patulin (PAT) is one of the mycotoxins commonly found in agricultural products and fruits, and has obvious toxic effects on animals and humans. PAT has been found to cause myocardial toxicity and oxidative damage, but the mechanism of myocardial toxicity remained to be elucidated. We investigated the toxic effects and potential mechanisms of PAT on human cardiomyocytes and explored the effects of reactive oxygen species (ROS) on them. The study showed that treatment with PAT for 24 h decreased cell viability and superoxide dismutase (SOD) activity, and increased ROS and lactate dehydrogenase (LDH) levels. Moreover, in addition to detecting increased γ-H2AX expression and observing nuclear damage, the comet assay also showed increased DNA tail distance in the PAT-treated group, followed by an increase in phosphorylation of the p53 protein and p21 protein expression, and a decrease in CDK1 and Cyclin B1 protein expression, and G2/M phase arrest. In addition, PAT induced endoplasmic reticulum stress (ERS) and induced apoptosis, as evidenced by Ca2+ increase, ER enlargement and swelling, and upregulation of ERS-related genes and proteins expression, and increased expression of three apoptotic pathway proteins under ERS, including CHOP, JNK, and caspase-12. Meanwhile, N-acetylcysteine (NAC, a ROS scavenger) reversed the negative effects of PAT treatment on cells. These results clarify that excessive ROS production by PAT-treated AC16 cells not only causes DNA damage, leading to cell cycle arrest, but also causes ERS, which triggers apoptotic pathways to cause apoptosis.

Infection of postharvest pear by Penicillium expansum is facilitated by the glycoside hydrolase (eglB) gene.

The primary reason for postharvest loss is blue mold disease which is mainly caused by Penicillium expansum. Strategies for disease control greatly depend on the understanding of mechanisms of pathogen-fruit interaction. A member of the glycoside hydrolase family, ß-glucosidase 1b (eglB), in P. expansum was significantly upregulated during postharvest pear infection. Glycoside hydrolases are a large group of enzymes that can degrade plant cell wall polymers. High homology was found between the glycoside hydrolase superfamily in P. expansum. Functional characterization and analysis of eglB were performed via gene knockout and complementation analysis. Although eglB deletion had no notable effect on P. expansum colony shape or microscopic morphology, it did reduce the production of fungal hyphae, thereby reducing P. expansum's sporulation and patulin (PAT) accumulation. Moreover, the deletion of eglB (ΔeglB) reduced P. expansum pathogenicity in pears. The growth, conidia production, PAT accumulation, and pathogenicity abilities of ΔeglB were restored to that of wild-type P. expansum by complementation of eglB (ΔeglB-C). These findings indicate that eglB contributes to P. expansum's development and pathogenicity. This research is a contribution to the identification of key effectors of fungal pathogenicity for use as targets in fruit safety strategies.

Synergistic toxicity induced by the co-exposure of tenuazonic acid and patulin in Caenorhabditis elegans: Daf-16 plays an important regulatory role.

Tenuazonic acid (TeA) and patulin (PAT), as the naturally occurring mycotoxins with various toxic effects, are often detected in environment and food chain, has attracted more and more attention due to their widespread and high contaminations as well as the coexistence, which leads to potential human and animals' risks. However, their combined toxicity has not been reported yet. In our study, C. elegans was used to evaluate the type of combined toxicity caused by TeA+PAT and its related mechanisms. The results showed that TeA and PAT can induce synergistic toxic effects based on Combination Index (CI) evaluation model (Chou-Talalay method), that is, the body length, brood size as well as the levels of ROS, CAT and ATP were significantly affected in TeA+PAT-treated group compared with those in TeA- or PAT-treated group. Besides, the expressions of oxidative (daf-2, daf-16, cyp-35a2, ctl-1, ctl-3, pmk-1, jnk-1, skn-1) and intestinal (fat-5, pod-2, egl-8, pkc-3, ajm-1, nhx-2) stress-related genes were disrupted, among which daf-16 displayed the most significant alternation. Further study on daf-16 gene defective C. elegans showed that the damages to the mutant nematodes were significantly attenuated. Since daf-2, daf-16, jnk-1 and pmk-1 are evolutionarily conserved, our findings could hint synergistic toxic effects of TeA+PAT on higher organisms.

Novel genetic tools improve Penicillium expansum patulin synthase production in Aspergillus niger.

Since the first CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system was developed for creating double-stranded DNA breaks, it has been adapted and improved for different biotechnological applications. In this issue of The FEBS Journal, Arentshorst et al. developed a novel approach to enhance transgene expression of a specific protein, patulin synthase (PatE) from Penicillium expansum, in the important industrial filamentous fungus Aspergillus niger. Their technique involved the disruption of selected genes with counter-effects on targeted protein production and simultaneous integration of glucoamylase landing sites into the disrupted gene locus such as protease regulator (prtT) in an ATP-dependent DNA helicase II subunit 1 (kusA or ku70)-deletion strain. Multiple copies of the PatE transgene expression cassette were introduced by CRISPR-Cas9-mediated insertion. The purified PatE was further used for structural and functional studies, and the technique laid the foundation for elevating the overall production of various proteins or chemicals in those industrially important fungi.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum.

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

Patulin alleviates hepatic lipid accumulation by regulating lipogenesis and mitochondrial respiration.

AIMS: The aim of this study is to evaluate the effects of patulin on hepatic lipid metabolism and mitochondrial oxidative function and elucidate the underlying molecular mechanisms. MAIN METHODS: The effects of patulin on hepatic lipid accumulation were evaluated in free fatty acid-treated AML12 or HepG2 cells through oil red O staining, triglyceride assay, real-time polymerase chain reaction, and western blotting. Alteration of mitochondrial oxidative capacity by patulin treatment was determined using Seahorse analysis to measure the oxygen consumption rate. KEY FINDINGS: The increased amounts of lipid droplets induced by free fatty acids were significantly reduced by patulin treatment. Patulin markedly activated the CaMKII/AMP-activated protein kinase (AMPK)/proliferator-activated receptor-γ coactivator (PGC)-1α signaling pathway in hepatocytes, reduced the expression of sterol regulatory element binding protein 1c (SREBP-1c) and lipogenic genes, and increased the expression of genes related to mitochondrial fatty acid oxidation. In addition, patulin treatment enhanced the mitochondrial consumption rate and increased the expression of mitochondrial oxidative phosphorylation proteins in HepG2 hepatocytes. The effects of patulin on anti-lipid accumulation; SREBP-1c, PGC-1α, and carnitine palmitoyltransferase 1 expression; and mitochondrial oxidative capacity were significantly prevented by compound C, an AMPK inhibitor. SIGNIFICANCE: Patulin is a potent inducer of the AMPK pathway, and AMPK-mediated mitochondrial activation is required for the efficacy of patulin to inhibit hepatic lipid accumulation. This study is the first to report that patulin is a promising bioactive compound that prevents the development and worsening of fatty liver diseases, including non-alcoholic fatty liver disease, by improving mitochondrial quality and lipid metabolism.

Structure elucidation and characterization of patulin synthase, insights into the formation of a fungal mycotoxin.

Patulin synthase (PatE) from Penicillium expansum is a flavin-dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in fruit and fruit-derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. This confirmed that PatE is active not only on the proposed patulin precursor ascladiol but also on several aromatic alcohols including 5-hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl-alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin.

Role of thiols and ascladiol production in patulin degradation by lactobacilli.

Patulin is a mycotoxin contaminant in various foods with apple products being its major dietary source. Yeast can reduce patulin levels during fermentation via biotransformation and thiol-adduct formation, with the ability of patulin to react with thiols being well known. Conversion of patulin to ascladiol by lactobacilli has been sparsely reported, while the contribution of thiols in reduction of patulin levels by lactobacilli remains undocumented. In this study, 11 strains of lactobacilli were screened for ascladiol formation in apple juice fermentation. Highest bioconversion was obtained for Lactiplantibacillus plantarum strains followed by Levilactobacillus brevis TMW1.465. Ascladiol production was also detected in several other lactobacilli species albeit in trace amounts. Reduction in patulin levels by Fructilactobacillus sanfranciscensis DMS 20451 and its glutathione reductase (ΔgshR) negative mutant was also assayed to determine the contribution of thiols. The hydrocinnamic acid reductase of Furfurilactobacillus milii did not contribute to reduction of patulin levels. In conclusion, this study demonstrated the potential of various lactobacilli in reduction of patulin levels via biotransformation of patulin to ascladiol, while also providing evidence for the role of thiol formation by lactobacilli and its presence in reducing patulin levels during fermentation.

Degradation of Patulin in Pear Juice and Apple Juice by Ascorbic Acid and the Combination of Ascorbic Acid and Ferrous Iron.

Patulin (PAT) is a toxic secondary metabolite produced by certain species of Penicillium sp. and Aspergillus sp. on apples and pears. In this study, we investigated the effects of ascorbic acid and the combination of ascorbic acid and ferrous iron on degradation of PAT in 100% pure pear juice and apple juice using high-performance liquid chromatography UV detector (HPLC-UVD). The addition of 2 different levels of ascorbic acid (143 or 286 µg/mL) into pear juice or apple juice containing 0.08 or 0.4 µg/mL of PAT showed 87.7-100% and 67.3-68.7% of PAT degradation rates, respectively, after 24 h incubation at 25 °C. Moreover, the addition of both ascorbic acid (143 or 286 µg/mL) and ferrous iron (0.033 or 0.11 µmol/mL) into pear juice or apple juice containing the same level of PAT exhibited higher PAT degradation rates (100 and 75-94%, respectively) than the addition of only ascorbic acid after 24 h incubation at 25 °C. Our data demonstrated that ascorbic acid plus ferrous iron as well as ascorbic acid were highly effective on degradation of PAT in pear juice and apple juice and that addition of both ascorbic acid and ferrous iron produced higher PAT degradation rates than addition of only ascorbic acid.

Insights into the Metabolic Response of Lactiplantibacillus plantarum CCFM1287 upon Patulin Exposure.

Patulin (PAT) is a common mycotoxin in the food industry, and is found in apple products in particular. Consumption of food or feed contaminated with PAT can cause acute or chronic toxicity in humans and animals. Lactiplantibacillus plantarum CCFM1287 is a probiotic strain that effectively degrades PAT in PBS and food systems. In this study, it was found that the concentration of PAT (50 mg/L) in MRS medium decreased by 85.09% during the first stages of CCFM1287 growth, and this change was consistent with the first-order degradation kinetic model. Meanwhile, the regulation of oxidative stress by L. plantarum CCFM1287 in response to PAT exposure and metabolic changes that occur during PAT degradation were investigated. The degree of intracellular damage was attenuated after 16 h of exposure compared to 8 h. Meanwhile, metabolomic data showed that 30 and 29 significantly different metabolites were screened intracellularly in the strain after 8 h and 16 h of PAT stress at 50 mg/L, respectively. The results of pathway enrichment analysis suggested that the purine metabolic pathway was significantly enriched at both 8 h and 16 h. However, as is consistent with the performance of the antioxidant system, the changes in Lactiplantibacillus diminished with increasing time of PAT exposure. Therefore, this study helps to further explain the mechanism of PAT degradation by L. plantarum CCFM1287.

Possible Reaction Mechanisms Involved in Degradation of Patulin by Heat-Assisted Cysteine under Highly Acidic Conditions.

Patulin (PAT) is one of mycotoxins that usually contaminates apple juice, and it is not easily detoxified by cysteine (CYS) at room temperature due to the highly acidic conditions based on the Michael addition reaction. However, it could be effectively degraded by a heating treatment at 120 °C for 30 min in the presence of cysteine. In our study, a total of eight degradation products (DP A-H) were characterized and identified via liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) in a negative ion mode, and their structures and formulas were proposed based on their accurate mass data. The fragmentation patterns of PAT and its degradation products were obtained from the MS/MS analysis. Meanwhile, the possible reaction mechanisms involved in the degradation of PAT were established and explained for the first time. According to the relation between the structure and toxicity of PAT, it could be deduced that the toxic effects of PAT degradation products were potentially much less than those of PAT-self.

Structure-based rational design of a short-chain dehydrogenase/reductase for improving activity toward mycotoxin patulin.

Patulin is a fatal mycotoxin that is widely detected in drinking water and fruit-derived products contaminated by diverse filamentous fungi. CgSDR from Candida guilliermondii represents the first NADPH-dependent short-chain dehydrogenase/reductase that catalyzes the reduction of patulin to the nontoxic E-ascladiol. To elucidate the catalytic mechanism of CgSDR, we solved its crystal structure in complex with cofactor and substrate. Structural analyses indicate that patulin is situated in a hydrophobic pocket adjacent to the cofactor, with the hemiacetal ring orienting toward the nicotinamide moiety of NADPH. In addition, we conducted structure-guided engineering to modify substrate-binding residue V187 and obtained variant V187F, V187K and V187W, whose catalytic activity was elevated by 3.9-, 2.2- and 1.7-fold, respectively. The crystal structures of CgSDR variants suggest that introducing additional aromatic stacking or hydrogen-bonding interactions to bind the lactone ring of patulin might account for the observed enhanced activity. These results illustrate the catalytic mechanism of SDR-mediated patulin detoxification for the first time and provide the upgraded variants that exhibit tremendous potentials in industrial applications.

Characterization of Two Dehydrogenases from Gluconobacter oxydans Involved in the Transformation of Patulin to Ascladiol.

Patulin is a mycotoxin that primarily contaminate apples and apple products. Whole cell or cell-free extracts of Gluconobacter oxydans ATCC 621 were able to transform patulin to E-ascladiol. Proteins from cell-free extracts were separated by anion exchange chromatography and fractions with patulin transformation activity were subjected to peptide mass fingerprinting, enabling the identification of two NADPH dependent short chain dehydrogenases, GOX0525 and GOX1899, with the requisite activity. The genes encoding these enzymes were expressed in E. coli and purified. Kinetic parameters for patulin reduction, as well as pH profiles and thermostability were established to provide further insight on the potential application of these enzymes for patulin detoxification.

Patulin Detoxification by Recombinant Manganese Peroxidase from Moniliophthora roreri Expressed by Pichia pastoris.

The fungal secondary metabolite patulin is a mycotoxin widespread in foods and beverages which poses a serious threat to human health. However, no enzyme was known to be able to degrade this mycotoxin. For the first time, we discovered that a manganese peroxidase (MrMnP) from Moniliophthora roreri can efficiently degrade patulin. The MrMnP gene was cloned into pPICZα(A) and then the recombinant plasmid was transformed into Pichia pastoris X-33. The recombinant strain produced extracellular manganese peroxidase with an activity of up to 3659.5 U/L. The manganese peroxidase MrMnP was able to rapidly degrade patulin, with hydroascladiol appearing as a main degradation product. Five mg/L of pure patulin were completely degraded within 5 h. Moreover, up to 95% of the toxin was eliminated in a simulated patulin-contaminated apple juice after 24 h. Using Escherichia coli as a model, it was demonstrated that the deconstruction of patulin led to detoxification. Collectively, these traits make MrMnP an intriguing candidate useful in enzymatic detoxification of patulin in foods and beverages.

Cytotoxicity of Mycotoxins and Their Combinations on Different Cell Lines: A Review.

Mycotoxins are secondary metabolites of molds and mainly produced by species of the genera Aspergillus, Penicillium and Fusarium. They can be synthesized on the field, during harvest as well as during storage. They are fairly stable compounds and difficult to remove. Among several hundreds of mycotoxins, according to the WHO, ochratoxin A, aflatoxins, zearalenone, deoxynivalenol, patulin, fumonisins as well as T-2 and HT-2 toxins deserve special attention. Cytotoxicity is one of the most important adverse properties of mycotoxins and is generally assessed via the MTT assay, the neutral red assay, the LDH assay, the CCK-8 assay and the ATP test in different cell lines. The apoptotic cell ratio is mainly assessed via flow cytometry. Aside from the assessment of the toxicity of individual mycotoxins, it is important to determine the cytotoxicity of mycotoxin combinations. Such combinations often exhibit stronger cytotoxicity than individual mycotoxins. The cytotoxicity of different mycotoxins often depends on the cell line used in the experiment and is frequently time- and dose-dependent. A major drawback of assessing mycotoxin cytotoxicity in cell lines is the lack of interaction typical for complex organisms (for example, immune responses).

Penicillium expansum Impact and Patulin Accumulation on Conventional and Traditional Apple Cultivars.

Penicillium expansum is a necrotrophic plant pathogen among the most ubiquitous fungi disseminated worldwide. It causes blue mould rot in apples during storage, transport and sale, threatening human health by secreting patulin, a toxic secondary metabolite that contaminates apples and apple-derived products. Nevertheless, there is still a lack of sufficient data regarding the resistance of different apple cultivars to P. expansum, especially ancient ones, which showed to possess certain resistance to plant diseases. In this work, we investigated the polyphenol profile of 12 traditional and 8 conventional apple cultivar and their resistance to P. expansum CBS 325.48. Eight polyphenolic compounds were detected; the most prominent were catechin, epicatechin and gallic acid. The highest content of catechin was detected in 'Apistar'-91.26 mg/100 g of fresh weight (FW), epicatechin in 'Bobovac'-67.00 mg/100 g of FW, and gallic acid in 'Bobovac' and 'Kraljevcica'-8.35 and 7.40 mg/100 g of FW, respectively. The highest content of patulin was detected in 'Kraljevcica' followed by 'Apistar'-1687 and 1435 µg/kg, respectively. In apple cultivars 'Brcko', 'Adamcica' and 'Idared', patulin was not detected. Furthermore, the patulin content was positively correlated with gallic acid (r = 0.4226; p = 0.002), catechin (r = 0.3717; p = 0.008) and epicatechin (r = 0.3305; p = 0.019). This fact indicates that higher contents of gallic acid, catechin and epicatechin negatively affected and boost patulin concentration in examined apple cultivars. This can be related to the prooxidant activity of polyphenolic compounds and sensitivity of P. expansum to the disturbance of oxidative status.

Thermal Stability and Degradation Kinetics of Patulin in Highly Acidic Conditions: Impact of Cysteine.

The thermal stability and degradation kinetics of patulin (PAT, 10 µmol/L) in pH 3.5 of phosphoric-citric acid buffer solutions in the absence and presence of cysteine (CYS, 30 µmol/L) were investigated at temperatures ranging from 90 to 150 °C. The zero-, first-, and second-order models and the Weibull model were used to fit the degradation process of patulin. Both the first-order kinetic model and Weibull model better described the degradation of patulin in the presence of cysteine while it was complexed to simulate them in the absence of cysteine with various models at different temperatures based on the correlation coefficients (R2 > 0.90). At the same reaction time, cysteine and temperature significantly affected the degradation efficiency of patulin in highly acidic conditions (p < 0.01). The rate constants (kT) for patulin degradation with cysteine (0.0036-0.3200 µg/L·min) were far more than those of treatments without cysteine (0.0012-0.1614 µg/L·min), and the activation energy (Ea = 43.89 kJ/mol) was far less than that of treatment without cysteine (61.74 kJ/mol). Increasing temperature could obviously improve the degradation efficiency of patulin, regardless of the presence of cysteine. Thus, both cysteine and high temperature decreased the stability of patulin in highly acidic conditions and improved its degradation efficiency, which could be applied to guide the detoxification of patulin by cysteine in the juice processing industry.