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Short Communication |
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1 Department of Soil Science and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI, USA
2 Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA
Correspondence
Joel A. Pedersen
joelpedersen{at}wisc.edu
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4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals.
These authors contributed equally to this work. 
Present address: Dipartimento di Scienze del Suolo della Pianta dell'Ambiente e delle Produzioni Animali, Università Federico II, Portici (NA), Italy.
Present address: US Geological Survey, Biological Resources Division, National Wildlife Health Center, Madison, WI, USA.
Three supplementary figures are available with the online version of this paper.
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). Scrapie and CWD differ from other TSEs in that epizootics can be maintained by horizontal transmission from infected to naïve animals (Hoinville, 1996; Miller & Williams, 2003) and be mediated by an environmental reservoir of infectivity (Greig, 1940; Miller & Williams, 2003). Infection of naïve sheep with scrapie following exposure to potentially contaminated environments is well established (Greig, 1940; Pálsson, 1979; Georgsson et al., 2006) and experiments with mule deer suggest a similar mode of transmission is possible with CWD (Miller et al., 2004).
The lack of clear evidence for vector-mediated TSE transmission prompted investigation of soil as a reservoir of prion infectivity (Schramm et al., 2006). Prions can persist in soils for years (Seidel et al., 2007; Brown & Gajdusek, 1991) and binding of PrPTSE by soil particles may maintain prions near the soil surface, thereby increasing animal exposure (Johnson et al., 2006; Cooke et al., 2007; Ma et al., 2007). Soil particle-associated agents are infectious orally (Seidel et al., 2007; Johnson et al., 2007). Prion sorption to some types of soil particles enhances oral TSE transmission (Johnson et al., 2007), providing an explanation for disease transmission despite presumably low levels of prions in soil environments.
Soils comprise complex mixtures of inorganic and organic constituents, and soil properties vary considerably across multiple spatial scales. The influence of soils on prion fate and environmental TSE transmission is expected to be complex, and abiotic soil components may affect the stability of prions present in soil. For example, birnessite group manganese oxide (MnO2) minerals rank among the strongest oxidants in soils (EH0=1.29 V) (Bricker, 1965). Soils subjected to alternating reducing and oxidizing conditions, such as those occurring in seasonally waterlogged or poorly drained areas, typically contain the highest accumulations of manganese oxide minerals (Post, 1999; Tebo et al., 2004). Well-drained soils may also contain these minerals due to previous wet conditions (McKenzie, 1989). Manganese III and IV minerals can mediate the transformation of numerous organic compounds, including herbicides (Barrett & McBride, 2005) and antibiotics (Zhang & Huang, 2005; Rubert & Pedersen, 2006).
As an initial step towards understanding potential abiotic transformations of prions in soil, we investigated PrPTSE degradation by aqueous suspensions of 
-MnO2, a synthetic manganese oxide mineral equivalent to the birnessite-family mineral vernadite (Villalobos et al., 2003). Using the method described by Murray (1974), we synthesized a poorly crystalline manganese oxide resembling -MnO2 with an average Mn oxidation state of +3.94 (Gao, 2007). Brains from clinically affected Syrian hamsters experimentally infected with hamster-adapted transmissible mink encephalopathy agent (HY strain) were used as a source of TSE agent. Brain homogenate (BH), 10 % w/v, was prepared in ddH2O and PrPTSE was purified as described previously (Johnson et al., 2006). For experiments using protein misfolding cyclic amplification (PMCA), PrPTSE was sodium phosphotungstate (PTA)-precipitated from infected BH (Safar et al., 1998). Protein concentrations were determined using the DC protein assay (Bio-Rad).
PrPTSE was incubated with suspended -MnO2 under ambient O2 conditions at room temperature for the time periods indicated in the figure legends. Reactions were conducted in 20 mM sodium acetate (pH 4.0), except for experiments examining the effect of pH, in which solutions were buffered with 20 mM sodium acetate (pH 4.0 and 5.0), MES (pH 6.0) or HEPES (pH 7.0 and 8.0). Final sample volumes were 40 µl. Reactions were terminated by dissolving MnO2 with 25 µl 500 mM EDTA (pH 8.0) at 80 °C. Dissolving of the -MnO2 (using ascorbic acid, citric acid or EDTA) liberated proteins, facilitating measurement of remaining protein without affecting PrPTSE levels or detection (data not shown); subsequent experiments used EDTA. The possibility of dissolved -MnO2 interfering with PrPTSE detection was examined by dissolving the manganese oxide prior to addition of PrPTSE. After quenching reactions, the pH was stabilized by the addition of 5 µl 1 M Tris/HCl (pH 8.0) to samples. For SDS-PAGE/immunoblot analysis, 20 µl 10x sample buffer (Johnson et al., 2006) was added, and samples were heated for 10 min at 100 °C.
A modification of the method described by Saá et al. (2006) was used for PMCA detection of PrPTSE. Uninfected hamsters were perfused with PBS containing 1 mM EDTA; harvested brains were homogenized to 10 % (w/v) in PBS containing 150 mM NaCl, 1 % Triton X-100, 0.5 % digitonin and complete protease inhibitor cocktail (Roche), then clarified by centrifugation for 5 min at 850 g. Prior to PMCA, PTA-purified PrPTSE (50 µg ml–1) was incubated with 5.6 mg -MnO2 ml–1 (PrPTSE : -MnO2=1 : 110) or EDTA-dissolved -MnO2. Dilutions of all samples, in uninfected BH, were aliquoted into 96-well PCR plates then placed at 37 °C in a Misonix 3000 sonicator equipped with a microplate horn. Each of the 120 cycles consisted of 10 s sonication at 80 % power followed by 30 min incubation. Following PMCA, 50 µl sample was mixed with 50 µl 4 % N-lauroyl-sarcosine in PBS and incubated with 40 µg proteinase K ml–1 for 1 h at 37 °C. Proteinase K digestion was terminated by addition of 1 µl phenylmethylsulfonyl fluoride-saturated ethanol. Samples were prepared for immunoblotting by PTA-precipitation and resuspending pellets in 5x sample buffer with 1 % N-lauroyl-sarcosine.
Proteins were fractionated by SDS-PAGE (4–20 % gradient) or 10 % Bistris gel (Invitrogen) for PMCA experiments, transferred to polyvinyl difluoride membranes and immunoblotted with PrP-specific antibodies: mAb 3F4 (1 : 40 000 dilution) or full-length polyclonal antibody Rab 9, pool 2 (1 : 10 000). Detection was achieved with horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit immunoglobulins.
Following 16 h incubation with -MnO2, PrPTSE immunoreactivity declined in proportion to the amount of -MnO2 in suspension (Fig. 1a). At the highest -MnO2 concentration tested (5 mg ml–1; PrPTSE : -MnO2=1 : 200), PrPTSE levels decreased below the limit of immunoblotting detection. The unstructured N-terminal portion of PrPTSE is susceptible to degradation; N-terminal cleavage leaves a 27–30 kDa infectious core (Bolton et al., 1982). When lower -MnO2 concentrations were used, the PrP appeared similar in size to the untreated controls, suggesting that -MnO2 did not selectively cleave the N terminus (Fig. 1a). We increased the amount of PrPTSE in the reactions by approximately 10-fold (from 33 to 333 µg ml–1) to estimate the extent to which -MnO2 degrades larger amounts of protein (Fig. 1b) and found that 3 mg -MnO2 ml–1 was capable of degrading a considerable fraction of the protein (PrPTSE : -MnO2=1 : 90 to 1 : 9). As a control, -MnO2 was dissolved prior to incubation with PrPTSE. The amount of PrPTSE present in the dissolved manganese control was substantially larger than in the -MnO2 samples, approximately equal to the starting material (Fig. 1b). These data indicate that -MnO2, not Mn2+ and EDTA, was responsible for the decrease in immunoreactivity.
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-MnO2 influenced the extent of prion protein loss (Fig. 1c
). Exposure for 1 h to 1.5 or 2.6 mg -MnO2 ml–1 (PrPTSE : -MnO2=1 : 60 and 1 : 100) had a limited effect on PrPTSE, whereas 24 and 168 h exposures produced substantial declines. Comparable amounts of protein remained after 24 and 168 h exposure to 1.5 mg -MnO2 ml–1, suggesting no further reaction (Fig. 1c). Decreases in MnO2 reactivity toward organic molecules over the course of reactions have been noted previously (e.g. Rubert & Pedersen, 2006) and have been attributed to adsorption of reaction products to the oxide surface, shifts in surface site distribution towards less reactive sites or both (Klausen et al., 1997).
The most probable explanation for the -MnO2-dependent loss of PrPTSE immunoreactivity is degradation of the protein. Birnessite dissolving following the incubation with PrPTSE excludes the possibility that the decreases in immunoblotting signal are due to PrPTSE sorption to mineral surfaces. In the analyses described above, we used the monoclonal antibody (mAb) 3F4, directed against a single epitope on the hydrophobic core of the PrP molecule (residues 109–112). To ensure that the observed declines in PrPTSE were not due to -MnO2 affecting the 3F4 epitope, degradation experiments were repeated with 3F4 and a polyclonal antibody directed against full-length PrP (Rab 9, pool 2). After treatment of PrPTSE with -MnO2, no immunoreactivity with Rab 9, pool 2 remained and lower molecular mass breakdown products were absent (Supplementary Fig. S1, available in JGV Online). These data are consistent with the hypothesis that -MnO2 degrades PrPTSE by breaking the polypeptide backbone of the protein, but do not exclude the possibility that -MnO2 also alters amino acid side chains.
Organic molecule degradation by -MnO2 typically exhibits pronounced pH dependence (Stone & Morgan, 1984). We examined the -MnO2-mediated degradation of PrPTSE as a function of pH over the range relevant for most natural soils (pH 4–8) (Supplementary Fig. S2, available in JGV Online). Under the experimental conditions employed (25 µg PrPTSE ml–1 exposed to 3.8 mg -MnO2 ml–1 for 16 h, PrPTSE : -MnO2=1 : 150), PrPTSE levels dropped below the limit of detection when reactions were performed at pH 4 or 5, whereas substantial PrPTSE remained following reactions at pH6.
Pathogenic prion proteins may be released into the environment in saliva (Mathiason et al., 2006), excreta (Safar et al., 2008) or from decomposing animal tissue in a complex mixture of biomolecules (Miller et al., 2004). To evaluate the -MnO2-mediated degradation of PrPTSE in the presence of biological macromolecules, we assessed degradation of infected BH by -MnO2 by assaying both total residual protein and PrPTSE (Fig. 2). Following 16 h incubation with 0.4 mg -MnO2 ml–1, little protein was observed on Coomassie brilliant blue-stained gels, and incubation with 3.7 mg -MnO2 ml–1 decreased total protein to undetectable levels (detection limit 
100 ng protein) (Fig. 2a). Dissolving of -MnO2 prior to incubation with the BH had little effect on protein levels. The PrPTSE present in infected BH was also diminished by exposure to -MnO2 (Fig. 2b). Compared with experiments using preparations enriched in PrPTSE, more -MnO2 was needed to degrade the PrPTSE in BH (compare Figs 1a and 2b). This may be due to the reductive dissolution of -MnO2 as it reacts with other biomolecules in BH and/or fouling the oxide surface by adsorbed biomolecules.
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-MnO2-mediated PrPTSE degradation, we diluted PrPTSE starting material to the limit of immunoblotting detection (Johnson et al., 2006; Hinkley et al., 2008). A 200-fold dilution of starting material was still detectable on immunoblots (data not shown), thus samples exhibiting no detectable immunoreactivity, such as PrPTSE treated with 5 mg -MnO2 ml–1 (Fig. 1a), contain at least 200-fold less PrPTSE than the starting material. To further assess PrPTSE loss, PMCA was used to determine the amount of PrP converting activity remaining after -MnO2 treatment. PMCA sensitively detects prions by measuring the ability of PrPTSE in a sample to convert PrPC to a proteinase K-resistant form (Saá et al., 2006). Converting activity of the PTA-purified PrPTSE starting material was detectable over four 10-fold dilutions (Fig. 3). Conversion activity of PTA-purified PrPTSE is diminished relative to infected BH (Fig. 3 and Supplementary Fig. S3, available in JGV Online), possibly due to increased aggregation of the PTA-purified agent. When samples were incubated with 5.6 mg -MnO2 ml–1 (PrPTSE : -MnO2=1 : 110) and assayed by PMCA, no converting activity was observed (Fig. 3). The limit of detection for PMCA is defined by the dilutions of the PTA-purified starting material (see above), indicating that 5.6 mg -MnO2 ml–1 decreased converting activity by at least four orders of magnitude.
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; Rao et al., 2007). Treatment of BH with -MnO2 (Fig. 3) indicates that protein degradation is not specific to PrPTSE; -MnO2 degrades most, if not all, brain proteins.
Birnessite-mediated PrPTSE degradation exhibited pronounced pH-dependence (Supplementary Fig. S2). Two non-mutually exclusive factors may have contributed to this behaviour. First, MnO2 redox potential increases and surface charge becomes less negative as pH declines (Bricker, 1965). Second, solution pH may influence the degree of protein sorption to MnO2 surfaces. The point of zero charge for -MnO2 occurs near pH 2.3 (Murray, 1974); -MnO2 particles carried a net negative charge at all pH values examined. Protein attachment to negatively charged surfaces is often maximal at the isoelectric point (pI) of the protein and declines when pH>pI due to repellent electrostatic interactions (Quiquampoix et al., 2002). The apparent average pI of PrPTSE aggregates is 4.6 (Ma et al., 2007); prion protein aggregates carried no net charge around this pH value. In most models of degradation of organic molecules by MnO2, sorption to the oxide surface represents a critical initial step (Stone, 1987). The increase in degradation near the apparent pI of PrPTSE aggregates is consistent with the importance of protein sorption to -MnO2 in the overall protein degradation process.
Our data suggest that manganese oxides in soils may promote PrPTSE inactivation, thereby reducing the probability of environmental TSE transmission. Previous investigation of soil manganese contributing to TSE development focused on dietary manganese and copper imbalance (Chihota et al., 2004; Gudmundsdottir et al., 2006; Ragnarsdottir & Hawkins, 2006; McBride, 2007). Attempts to correlate soil or forage manganese and copper concentrations with TSE incidence have met with limited success. Future attempts to link TSE incidence with soil manganese levels should examine the mineral form of the element in addition to total (or bioavailable) manganese concentration.
Prion fate in terrestrial environments probably depends on soil composition. Abiotic prion degradation could be expected in soil environments rich in MnO2, including young, and currently or formerly poorly drained soils (Post, 1999; Tebo et al., 2004). Our results indicate acidic soil conditions may also promote MnO2-mediated PrPTSE degradation. Under the experimental conditions employed, -MnO2-mediated PrPTSE degradation occurs over relatively short periods (Fig. 1c) and reduces PrPTSE-converting activity by at least a factor of 104 (Fig. 3). Our findings also suggest that MnO2 may be effective as a reactive burial material in the disposal of prion-infected materials. Use of MnO2 for the decontamination of prion-contaminated soils also warrants investigation.
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ACKNOWLEDGEMENTS |
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-MnO2 preparation. We gratefully acknowledge two anonymous reviewers for their constructive comments. This work was supported in part by NSF CAREER award BES-0547484 (J. A. P), USEPA grant 4C-R070-NAEX (J. A. P.) and DOD grants DAMD17-03-1-0369 (J. M. A.) and DAMD17-03-1-0294 (D. M.). |
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Received 17 April 2008;
accepted 14 August 2008.
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