Deepoxy-deoxynivalenol retains some immune-modulatory properties of the parent molecule deoxynivalenol in piglets.

Deoxynivalenol (DON) is the most abundant trichothecene in food and feed. It causes both acute and chronic disorders of the human and animal intestine, liver and the immune system. The structural basis for the toxicity of DON has not been fully elucidated. Using the pig as a target and a model species for human, the toxicity of DON and its deepoxy-metabolite (DOM-1) was compared. Animals were exposed by gavage to 1 and 0.5 nmol toxin/kg b.w./day for 2 and 3 weeks respectively. Whatever the dose/duration, DOM-1 was less toxic than DON in terms of weight gain and emesis. In the 3-week experiment, animals were vaccinated with ovalbumin, and their immune response was analyzed in addition to tissue morphology, biochemistry and hematology. DON impaired the morphology of the jejunum and the ileum, reduced villi height, decreased E-cadherin expression and modified the intestinal expression of cytokines. Similarly, DON induced hepatotoxicity as indicated by the lesion score and the blood biochemistry. By contrast, DOM-1 only induced minimal intestinal toxicity and did not trigger hepatotoxicity. As far as the immune response was concerned, the effects of ingesting DOM-1 were similar to those caused by DON, as measured by histopathology of lymphoid organs, PCNA expression and the specific antibody response. Taken together, these data demonstrated that DOM-1, a microbial detoxification product of DON, was not toxic in the sensitive pig model but retained some immune-modulatory properties of DON, especially its ability to stimulate a specific antibody response during a vaccination protocol.

Intestinal toxicity of the masked mycotoxin deoxynivalenol-3-ß-D-glucoside.

Natural food contaminants such as mycotoxins are an important problem for human health. Deoxynivalenol (DON) is one of the most common mycotoxins detected in cereals and grains. Its toxicological effects mainly concern the immune system and the gastrointestinal tract. This toxin is a potent ribotoxic stressor leading to MAP kinase activation and inflammatory response. DON frequently co-occurs with its glucosylated form, the masked mycotoxin deoxynivalenol-3-ß-D-glucoside (D3G). The toxicity of this later compound remains unknown in mammals. This study aimed to assess the ability of D3G to elicit a ribotoxic stress and to induce intestinal toxicity. The toxicity of D3G and DON (0-10 µM) was studied in vitro, on the human intestinal Caco-2 cell line, and ex vivo, on porcine jejunal explants. First, an in silico analysis revealed that D3G, contrary to DON, was unable to bind to the A-site of the ribosome peptidyl transferase center, the main targets for DON toxicity. Accordingly, D3G did not activate JNK and P38 MAPKs in treated Caco-2 cells and did not alter viability and barrier function on cells, as measured by the trans-epithelial electrical resistance. Treatment of intestinal explants for 4 h with 10 µM DON induced morphological lesions and up-regulated the expression of pro-inflammatory cytokines as measured by qPCR and pan-genomic microarray analysis. By contrast, expression profile of D3G-treated explants was similar to that of controls, and these explants did not show histomorphology alteration. In conclusion, our data demonstrated that glucosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity.

Rhodococcus erythropolis MTHt3 biotransforms ergopeptines to lysergic acid.

BACKGROUND: Ergopeptines are a predominant class of ergot alkaloids produced by tall fescue grass endophyte Neotyphodium coenophialum or cereal pathogen Claviceps purpurea. The vasoconstrictive activity of ergopeptines makes them toxic for mammals, and they can be a problem in animal husbandry. RESULTS: We isolated an ergopeptine degrading bacterial strain, MTHt3, and classified it, based on its 16S rDNA sequence, as a strain of Rhodococcus erythropolis (Nocardiaceae, Actinobacteria). For strain isolation, mixed microbial cultures were obtained from artificially ergot alkaloid-enriched soil, and provided with the ergopeptine ergotamine in mineral medium for enrichment. Individual colonies derived from such mixed cultures were screened for ergotamine degradation by high performance liquid chromatography and fluorescence detection. R. erythropolis MTHt3 converted ergotamine to ergine (lysergic acid amide) and further to lysergic acid, which accumulated as an end product. No other tested R. erythropolis strain degraded ergotamine. R. erythropolis MTHt3 degraded all ergopeptines found in an ergot extract, namely ergotamine, ergovaline, ergocristine, ergocryptine, ergocornine, and ergosine, but the simpler lysergic acid derivatives agroclavine, chanoclavine, and ergometrine were not degraded. Temperature and pH dependence of ergotamine and ergine bioconversion activity was different for the two reactions. CONCLUSIONS: Degradation of ergopeptines to ergine is a previously unknown microbial reaction. The reaction end product, lysergic acid, has no or much lower vasoconstrictive activity than ergopeptines. If the genes encoding enzymes for ergopeptine catabolism can be cloned and expressed in recombinant hosts, application of ergopeptine and ergine degrading enzymes for reduction of toxicity of ergot alkaloid-contaminated animal feed may be feasible.

Deoxynivalenol (DON) sulfonates as major DON metabolites in rats: from identification to biomarker method development, validation and application.

Deoxynivalenol (DON) is a trichothecene mycotoxin regularly occurring in cereals. Rats are often used to study toxicokinetics of DON and related compounds, yet only about 30 % of the administered dose is typically recovered. Recently, it was reported that DON is partly metabolised to previously undetected DON- and deepoxy-DON (DOM) sulfonate in rats and tentative structures were proposed. The present work describes the production and characterisation of DON-, DOM- and DON-3-glucoside (D3G) sulfonates of three different series; the development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS)-based methods for determination of DON, DOM, D3G and their sulfonates in rat faeces and urine; and application of the methods to samples from a DON and D3G feeding trial with rats. In addition to previously produced DON sulfonates (DONS) 1, 2 and 3, D3G sulfonates 1, 2 and 3; and DOM sulfonates (DOMS) 2 and 3 were synthesised, purified and characterised. The developed methods showed apparent recoveries of all investigated compounds between 68 and 151 % in faeces and between 48 and 113 % in urine. The recovery of DON, D3G and their metabolites from faeces and urine of rats (n = 6) administered in a single dose of 2.0 mg/kg b.w. DON or the equimolar amount of D3G was 75 ± 9 % for the DON group and 68 ± 8 % for the D3G group. DON-, DOM- and D3G sulfonates excreted in faeces accounted for 48 and 47 % of the total amount of administered DON and D3G. Urinary excretion of sulfonates was <1 %. In both treatment groups, DONS 2 was the major metabolite 0-24 h after treatment, whereas DOMS 2 was predominant thereafter. The developed methods can also be used for investigation of DON (conjugate) sulfonate formation in other animal species.

Bacterial Lactonases ZenA with Noncanonical Structural Features Hydrolyze the Mycotoxin Zearalenone.

Zearalenone (ZEN) is a mycoestrogenic polyketide produced by Fusarium graminearum and other phytopathogenic members of the genus Fusarium. Contamination of cereals with ZEN is frequent, and hydrolytic detoxification with fungal lactonases has been explored. Here, we report the isolation of a bacterial strain, Rhodococcus erythropolis PFA D8-1, with ZEN hydrolyzing activity, cloning of the gene encoding α/ß hydrolase ZenA encoded on the linear megaplasmid pSFRL1, and biochemical characterization of nine homologues. Furthermore, we report site-directed mutagenesis as well as structural analysis of the dimeric ZenARe of R. erythropolis and the more thermostable, tetrameric ZenAScfl of Streptomyces coelicoflavus with and without bound ligands. The X-ray crystal structures not only revealed canonical features of α/ß hydrolases with a cap domain including a Ser-His-Asp catalytic triad but also unusual features including an uncommon oxyanion hole motif and a peripheral, short antiparallel ß-sheet involved in tetramer interactions. Presteady-state kinetic analyses for ZenARe and ZenAScfl identified balanced rate-limiting steps of the reaction cycle, which can change depending on temperature. Some new bacterial ZEN lactonases have lower KM and higher kcat than the known fungal ZEN lactonases and may lend themselves to enzyme technology development for the degradation of ZEN in feed or food.

Chronic ingestion of deoxynivalenol and fumonisin, alone or in interaction, induces morphological and immunological changes in the intestine of piglets.

Deoxynivalenol (DON) and fumonisins (FB) are mycotoxins produced by Fusarium species, which naturally co-occur in animal diets. The gastrointestinal tract represents the first barrier met by exogenous food/feed compounds. The purpose of the present study was to investigate the effects of DON and FB, alone and in combination, on some intestinal parameters, including morphology, histology, expression of cytokines and junction proteins. A total of twenty-four 5-week-old piglets were randomly assigned to four different groups, receiving separate diets for 5 weeks: a control diet; a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg); or both toxins. Chronic ingestion of these contaminated diets induced morphological and histological changes, as shown by the atrophy and fusion of villi, the decreased villi height and cell proliferation in the jejunum, and by the reduced number of goblet cells and lymphocytes. At the end of the experiment, the expression levels of several cytokines were measured by RT-PCR and some of them (TNF-α, IL-1ß, IFN-γ, IL-6 and IL-10) were significantly up-regulated in the ileum or the jejunum. In addition, the ingestion of contaminated diets reduced the expression of the adherent junction protein E-cadherin and the tight junction protein occludin in the intestine. When animals were fed with a co-contaminated diet (DON+FB), several types of interactions were observed depending on the parameters and segments assessed: synergistic (immune cells); additive (cytokines and junction protein expression); less than additive (histological lesions and cytokine expression); antagonistic (immune cells and cytokine expression). Taken together, the present data provide strong evidence that chronic ingestion of low doses of mycotoxins alters the intestine, and thus may predispose animals to infections by enteric pathogens.

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1.

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B(1) and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B(1). We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B(1). Pyruvate was found to be the preferred co-substrate and amino group receptor (K (M) = 490 µM at 10 µM hydrolyzed fumonisin B(1)) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 µM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K (M) = 1.1 µM and k (cat) = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.

An aminotransferase from bacterium ATCC 55552 deaminates hydrolyzed fumonisin B1.

Previous research identified several microorganisms and pathways capable of degrading the mycotoxin fumonisin B1 (FB1). Degradation of FB1 by microorganisms seems to comprise two essential steps: hydrolysis to hydrolyzed fumonisin B1 (HFB1) and deamination of the hydrolysis product. One of the previously studied microorganisms was the Gram negative bacterium ATCC 55552. The gene corresponding to the first step of FB1 degradation in this bacterium was identified, but the genetic basis for deamination of the hydrolyzed intermediate remained unexplained (Duvick et al. 2000, PCT patent application WO200004158). Here we report the sequence and HFB1-deaminating activity of a novel aminotransferase encoded by the bacterium ATCC 55552. The corresponding gene was identified, sequenced, and over-expressed in Escherichia coli. Cell lysates of the recombinant E. coli strain showed distinct HFB1-deaminating activity in the presence of pyridoxal phosphate and pyruvate, as was demonstrated by liquid chromatography-mass spectrometry. Thus, we suggest the novel enzyme to be part of the fumonisin catabolic pathway of the bacterium ATCC 55552.

Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B(1).

BACKGROUND: Fumonisin B(1) is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B(1), and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B(1). In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. RESULTS: When expressed from a T7 promoter at 30 degrees C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG) used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3). Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30 degrees C or less, but not at 37 degrees C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3), which co-expresses two cold-adapted chaperonins, at 11 degrees C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the binding buffer were performed to obtain 1.45 mg of apparently homogeneous aminotransferase per liter of expression culture. CONCLUSIONS: We found that only reduction of temperature, but not reduction of expression level or fusion to maltose-binding protein helped to produce correctly folded, active aminotransferase FumI in E. coli. Our results may provide a starting point for soluble expression of related aminotransferases or other aggregation-prone proteins in E. coli.

An In Silico Target Fishing Approach to Identify Novel Ochratoxin A Hydrolyzing Enzyme.

Ochratoxin A (OTA), a mycotoxin that is of utmost concern in food and feed safety, is produced by fungal species that mainly belong to the Aspergillus and Penicillium genera. The development of mitigation strategies to reduce OTA content along the supply chains is key to ensuring safer production of food and feed. Enzyme-based strategies are among the most promising methods due to their specificity, efficacy, and multi-situ applicability. In particular, some enzymes are already known for hydrolyzing OTA into ochratoxin alpha (OTα) and phenylalanine (Phe), eventually resulting in detoxification action. Therefore, the discovery of novel OTA hydrolyzing enzymes, along with the advancement of an innovative approach for their identification, could provide a broader basis to develop more effective mitigating strategies in the future. In the present study, a hybrid in silico/in vitro workflow coupling virtual screening with enzymatic assays was applied in order to identify novel OTA hydrolyzing enzymes. Among the various hits, porcine carboxypeptidase B was identified for the first time as an effective OTA hydrolyzing enzyme. The successful experimental endorsement of findings of the workflow confirms that the presented strategy is suitable for identifying novel OTA hydrolyzing enzymes, and it might be relevant for the discovery of other mycotoxin- mitigating enzymes.

Reduced toxicity of 3-epi-deoxynivalenol and de-epoxy-deoxynivalenol through deoxynivalenol bacterial biotransformation: In vivo analysis in piglets.

Ingestion of deoxynivalenol (DON), one of the most common mycotoxin contaminants of cereals, leads to adverse effects for animal and human health. Bacterial biotransformation is a strategy to mitigate the toxicity of this mycotoxin. The present study aims to evaluate the toxicity of two bacterial biotranformation products of DON: 3-epi-deoxynivalenol (3-epi-DON) and de-epoxy-deoxynivalenol (DOM-1) through zootechnical, hematological, histological and immunological assays. Twenty-four 4-weeks-old piglets received a control diet or a diet contaminated with 3 mg kg-1 DON, DOM-1, or 3-epi-DON for 7 days. Sample tissues were collected for histomorphometrical analysis, expression of cytokines and cell protein junctions. The zootechnical and hematological parameters were not modulated by any treatment. Ingestion of DON induced histological alterations in the intestine, liver and lymphoid organs, as well as an overexpression of pro-inflammatory cytokines, E-cadherin and occludin. These changes were not observed in piglets receiving the DOM-1 and 3-epi-DON contaminated diets. Pigs fed 3-epi-DON contaminated diet showed an increase in IgM levels in comparison with other diets, while no change was observed in IgA and IgG levels among the diets. Our results indicate that DOM-1 and 3-epi-DON are not toxic for piglets; thus bacterial biotransformation seems to be a sustainable alternative to reduce mycotoxin toxicity.

Detoxification of the Fumonisin Mycotoxins in Maize: An Enzymatic Approach.

Enzymatic detoxification has become a promising approach for control of mycotoxins postharvest in grains through modification of chemical structures determining their toxicity. In the present study fumonisin esterase FumD (EC 3.1.1.87) (FUMzyme®; BIOMIN, Tulln, Austria), hydrolysing fumonisin (FB) mycotoxins by de-esterification, was utilised to develop an enzymatic reduction method in a maize kernel enzyme incubation mixture. Efficacy of the FumD FB reduction method in "low" and "high" FB contaminated home-grown maize was compared by monitoring FB1 hydrolysis to the hydrolysed FB1 (HFB1) product utilising a validated LC-MS/MS analytical method. The method was further evaluated in terms of enzyme activity and treatment duration by assessing enzyme kinetic parameters and the relative distribution of HFB1 between maize kernels and the residual aqueous environment. FumD treatments resulted in significant reduction (≥80%) in "low" (≥1000 U/L, p < 0.05) and "high" (100 U/L, p < 0.05; ≥1000 U/L, p < 0.0001) FB contaminated maize after 1 h respectively, with an approximate 1:1 µmol conversion ratio of FB1 into the formation of HFB1. Enzyme kinetic parameters indicated that, depending on the activity of FumD utilised, a significantly (p < 0.05) higher FB1 conversion rate was noticed in "high" FB contaminated maize. The FumD FB reduction method in maize could find application in commercial maize-based practices as well as in communities utilising home-grown maize as a main dietary staple and known to be exposed above the tolerable daily intake levels.

MicroRNAs in porcine uterus and serum are affected by zearalenone and represent a new target for mycotoxin biomarker discovery.

The mycotoxin zearalenone (ZEN) poses a risk to animal health because of its estrogenic effects. Diagnosis of ZEN-induced disorders remains challenging due to the lack of appropriate biomarkers. In this regard, circulating microRNAs (small non-coding RNAs) have remarkable potential, as they can serve as indicators for pathological processes in tissue. Thus, we combined untargeted and targeted transcriptomics approaches to investigate the effects of ZEN on the microRNA expression in porcine uterus, jejunum and serum, respectively. To this end, twenty-four piglets received uncontaminated feed (Control) or feed containing 0.17 mg/kg ZEN (ZEN low), 1.46 mg/kg ZEN (ZEN medium) and 4.58 mg/kg ZEN (ZEN high). After 28 days, the microRNA expression in the jejunum remained unaffected, while significant changes in the uterine microRNA profile were observed. Importantly, 14 microRNAs were commonly and dose-dependently affected in both the ZEN medium and ZEN high group, including microRNAs from the miR-503 cluster (i.e. ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-503 and ssc-miR-542-3p). Predicted target genes for those microRNAs are associated with regulation of gene expression and signal transduction (e.g. cell cycle). Although the effects in serum were less pronounced, receiver operating characteristic analysis revealed that several microRNA ratios were able to discriminate properly between non-exposed and ZEN-exposed pigs (e.g. ssc-miR-135a-5p/ssc-miR-432-5p, ssc-miR-542-3p/ssc-miR-493-3p). This work sheds new light on the molecular mechanisms of ZEN, and fosters biomarker discovery.

Biotransformation of the Mycotoxin Zearalenone to its Metabolites Hydrolyzed Zearalenone (HZEN) and Decarboxylated Hydrolyzed Zearalenone (DHZEN) Diminishes its Estrogenicity In Vitro and In Vivo.

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.

Engineered bakers yeast as a sensitive bioassay indicator organism for the trichothecene toxin deoxynivalenol.

The aim of this study was to increase the sensitivity of Saccharomyces cerevisiae towards trichothecene toxins, in particular to deoxynivalenol (DON), in order to improve the utility of this yeast as a bioassay indicator organism. We report the construction of a strain with inactivated genes (PDR5, PDR10, PDR15) encoding ABC transporter proteins with specificity for the trichothecene deoxynivalenol, with inactivated AYT1 (encoding a trichothecene-3-O-acetyltransferase), and inactivated UBI4 and UBP6 genes. Inactivation of the stress inducible polyubiquitin gene UBI4 or the ubiquitin protease UBP6 increased DON sensitivity, the inactivation of both genes had a synergistic effect. The resulting pdr5 pdr10 pdr15 ayt1 ubp6 ubi4 mutant strain showed 50% growth inhibition at a DON concentration of 5 mg/l under optimal conditions. The development of a simple two step assay for microbial DON degradation in 96 well microtiter format and its testing with the DON detoxifying bacterium BBSH 797 is reported.

Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA.

Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10. This fusion neither interfered with procapsid assembly nor affected the morphology of the prolate-shaped procapsid. However, the pRNA binding and virion assembly activity were greatly reduced. Such decreased functions can be switched back on by the removal of the tag via protease cleavage, supporting the previous finding that the N-terminus of gp10 is essential for the pRNA binding. The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA. It is speculated that the fusion of the tag generated physical hindrance to pRNA binding, with greater influence for the dimers than the monomers due to their size. These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.

Grouping of ferritin and gold nanoparticles conjugated to pRNA of the phage phi29 DNA-packaging motor.

The bacteriophage phi29 DNA-packaging motor, which translocates and compresses the DNA genome of the phage into its procapsid during virion assembly, involves an essential ring formed by the packaging RNA (pRNA). We attached electron-dense nanoparticles to pRNA by hybridizing a DNA oligonucleotide with a biotin or thiol modification to a 3'-extension of core pRNA, and by coupling streptavidin and biotinylated ferritin, or 5 nm and 10 nm colloidal gold particles, to these modifications. The pRNA conjugates bound to RNA-free phi29 procapsids, and such nanoparticle-bearing procapsids were isolated by ultracentrifugation and analyzed. Electron microscopy showed that ferritin and gold particles were specifically attached to the side of the phi29 procapsid harboring the connector, which is the pRNA binding site. The pRNA-ferritin conjugates bound to procapsids with the same efficiency as pRNA which lacked the high molecular mass label. However, full DNA packaging efficiency in an in vitro phage assembly assay was only reached after the label of such isolated procapsid-pRNA complexes had been released with RNase H. The results provide approaches to assembled ferritin or gold-containing nano-complexes via pRNA mediated assembly.

Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29.

During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864-3871] suggested that the foothold for pRNA was the connector and that the pRNA-connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS-PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA-connector complex and that the foothold for pRNA is the connector but not the capsid protein.

Translocation of nicked but not gapped DNA by the packaging motor of bacteriophage phi29.

The biomolecular mechanism that the double-stranded DNA viruses employ to insert and package their genomic DNA into a preformed procapsid is still elusive. To better characterize this process, we investigated packaging of bacteriophage phi29 DNA with structural alterations. phi29 DNA was modified in vitro by nicking at random sites with DNase I, or at specific sites with nicking enzyme N.BbvC IA. Single-strand gaps were created by expanding site-specific nicks with T4 DNA polymerase. Packaging of modified phi29 DNA was studied in a completely defined in vitro system. Nicked DNA was packaged at full genome length and with the same efficiency as untreated DNA. Nicks were not repaired during packaging. Gapped DNA was packaged only as a fragment corresponding to the DNA between the genome terminus and gap. Thus the phi29 DNA packaging machinery tolerated nicks, but stopped at gaps. The packaging motor did not require a nick-free DNA backbone, but the presence of both DNA strands, for uninterrupted packaging.

Microbial biotransformation of DON: molecular basis for reduced toxicity.

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 µM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.