Influence of Prion Strain on Prion Protein Adsorption to Soil in a

Jun 16, 2009 - encephalopathy (BSE, or “mad cow” disease). Prion adsorption to soil may play an important role in prion mobility, proteolysis, and...
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Environ. Sci. Technol. 2009, 43, 5242–5248

Influence of Prion Strain on Prion Protein Adsorption to Soil in a Competitive Matrix SAMUEL E. SAUNDERS,† JASON C. BARTZ,‡ AND S H A N N O N L . B A R T E L T - H U N T * ,† Department of Civil Engineering, University of Nebraska-Lincoln, Peter Kiewit Institute, Omaha, Nebraska, and Department of Medical Microbiology and Immunology, Creighton University, Omaha, Nebraska

Received February 16, 2009. Revised manuscript received May 22, 2009. Accepted June 2, 2009.

It is likely that the soil environment serves as a stable reservoir of infectious chronic wasting disease (CWD) and scrapie prions, as well as a potential reservoir of bovine spongiform encephalopathy (BSE, or “mad cow” disease). Prion adsorption to soil may play an important role in prion mobility, proteolysis, and infectivity. Differences in PrP environmental fate are possible due to the strain- and species-dependent structure of PrPSc. Kinetic and isothermal studies of PrP adsorption to sand and two whole soils were conducted using HY and DY TME-infected hamster, uninfected hamster, and CWD-infected elk brain homogenates as competitive PrP sources. The role of the N-terminus in PrP adsorption was also investigated. We report strain and species differences in PrP adsorption to soil over time and as a function of aqueous concentration, indicating that the fate of prions in the environment may vary with the prion strain and species infected. Our data also provide evidence that the N-terminal region of PrP enhances adsorption to clay but may hinder adsorption to sand. PrP adsorption was maximal at an intermediate aqueous concentration, most likely due to the competitive brain homogenate matrix in which it enters the soil environment.

Introduction Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative diseases impacting a number of mammals, including cattle (bovine spongiform encephalopathy, BSE, or “mad cow” disease), sheep and goats (scrapie), deer, elk, and moose (chronic wasting disease, CWD), and humans (Creutzfeldt-Jakob disease (CJD) and others) (1). Strong evidence indicates that the infectious agent of prion diseases is solely PrPSc (the prion), an abnormally folded isoform of a normal cellular protein, PrPc (1, 2). The misfolded conformation of PrPSc conveys distinct biological and physicochemical properties, including resistance to proteolysis and inactivation techniques, increased hydrophobicity, and a propensity for aggregation (1, 3). Scrapie and CWD are horizontally transmissible and remain infectious after years in the environment (4). The CWD and scrapie agent is shed from living hosts and present in mortalities (4). The disposal of BSE mortalities, both in * Corresponding author e-mail: [email protected]. † University of Nebraska-Lincoln. ‡ Creighton University. 5242

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the past and during potential future outbreaks, serves as another environmental source of prions with the potential to infect humans. CWD and scrapie are orally transmissible (5, 6), and the nasal cavity is also an effective route of infection (7, 8). Prions bound to soil particles remain infectious through oral consumption (9, 10). The potential for CWD or scrapie transmission by exposure to contaminated soil is possible since cervids and ruminants are known to ingest and inhale large amounts of soil (11, 12). Thus, soil and soil minerals may act as significant environmental reservoirs of infectivity. Prions are known to adsorb to a wide range of soils and soil minerals, including quartz sand, organic matter, and clay minerals (4, 13). Prion adsorption is strongly irreversible and resistant to desorption by detergent and chaotropic treatments (14, 15). Purified PrPSc and recombinant PrP (recPrP) have maximal adsorption in less than 2 h (15-17), and previous isothermal studies have used equilibration times as short as 10 min and no longer than 24 h (17-19). However, when infectious brain homogenate is the prion source, maximum PrP adsorption occurs no earlier than 24 h for clay and after 30 days for quartz sand (13). Additionally, PrP adsorption capacities are 2-5 orders of magnitude lower than in comparable studies using recPrP or purified PrPSc. This suggests that the competitive matrix (e.g., animal tissue, blood, saliva, or excreta) in which prions enter the environment affects adsorption. Competition for sorption sites and interactions on mineral surfaces between prions and other proteins, carbohydrates, lipids, and nucleic acids have been implicated as significant factors in PrP adsorption (13). The three-dimensional structure of PrPSc is unknown; therefore, determination of specific mechanisms of PrPSc adsorption is challenging. The most significant adsorption mechanism of recPrP is electrostatic attraction between positively charged peptides and negatively charged mineral surfaces (15, 20). Since PrPSc is highly insoluble and aggregated, hydrophobic interactions could play a large role in PrPSc sorption to amorphous clay. In one isothermal study, adsorption of purified PrPSc to quartz sand increased linearly with aqueous PrP concentration (Cw), but no maximum was reached (18). Isotherms of recPrP adsorption to two clays had linear increases in adsorbed concentration (Cs) at low Cw followed by a maximum plateau (19). However, Pucci et al. found no linear phase with recPrP adsorption to two whole soils, with a plateau reached at near-zero Cw (17). The environmental fate of PrPSc may vary with strain and species. Prion strains are operationally defined by distinct neuropathological characteristics (21). The amino acid sequence of PrP is species specific, and PrPSc conformation is strain dependent (22-26). To date, differences in observed PrP adsorption as a function of prion strain or animal species have not been explored. The objectives of this research were to quantitatively compare prion adsorption to soil as a function of prion strain and species and investigate PrP adsorption under isothermal conditions. We hypothesize that the competitive matrix in which prions enter the environment can significantly affect prion interactions with soil, and that these interactions will vary with prion strain and species. In this report, a direct detection method previously developed by the authors was used to quantify prion adsorption (13). This method eliminates the need to desorb prions prior to detection and allows for quantitative measure of adsorbed prions, including a complete prion mass balance (solid plus aqueous fractions). 10.1021/es900502f CCC: $40.75

 2009 American Chemical Society

Published on Web 06/16/2009

Materials and Methods Tissue Sources. Brain tissue was collected from hamsters infected with the HY TME (hyper strain of transmissible mink encephalopathy (TME)) and DY TME (drowsy strain of TME) agents at terminal disease (29). Brain tissue from uninfected hamsters and an elk infected with CWD were also used (30). Brain tissue was homogenized to 10% (w/v) in Dulbecco’s phosphate-buffered saline (DPBS) without Ca2+ or Mg2+ (Mediatech, Herndon, VA) using strain dedicated Tenbroeck tissue grinders (Kontes, Vineland, NJ). For proteinase K (PK) digestion, tissue homogenates were digested with 25 µg per mL of PK (Roche Diagnostics Corporation, Indianapolis, IN) and were incubated at 37 °C under constant agitation for 1 h. PK digestion was stopped by the addition of Pefabloc (100 µg/µL, Roche Diagnostics, Penzberg, Germany). Batch Sorption Assays. Fine white sand (Fisher Scientific, Pittsburgh, PA), sandy loam soil, and silty clay loam soil, were used to study prion adsorption. Soil characteristics are reprinted from ref 13 in Tables S1 and S2. For batch kinetic and isotherm studies, brain homogenate was combined with 10 mg of fine sand, 3 mg of sandy loam, or 1 mg of silty clay loam in a total volume of 200 µL in DPBS in 0.2 mL PCR tubes (Fisher Scientific). All experiments were conducted at pH 7. The soil and brain homogenate mixtures were gently rotated at 24 rpm (Mini Labroller, Labnet, Edison, NJ) at 22 °C. Samples were removed at specified time points and allowed to settle or centrifuged at 100g for 5 s. For isothermal experiments, samples were equilibrated for 1 week (168 h) unless otherwise noted. The supernatant was removed and the pellets were washed 2 times with DPBS. The original supernatant, first wash, and final pellet were collected and stored at -80 °C. Subsequent washes did not contain measurable PrP (13). All batch kinetic and isotherm experiments were performed in triplicate. Direct Detection Assay. A direct detection method described previously (13) was used without modification to quantify prion adsorption to soil. This method combines the direct detection method developed by Genovesi et al. (27) with the 96-well immunoassay method developed by Kramer and Bartz (28) to quantify PrP adsorbed to soil and minerals. Briefly, soil pellets were saturated with bovine serum albumin (Sigma Aldrich, St. Louis, MO), then incubated with 3 M guanidinium thiocyanate (Sigma Aldrich), and washed with DPBS. No detectable PrP was desorbed by the guanidinium treatment (data not shown). Soil pellets were blocked with blotto (Biorad, Hercules, CA) and incubated with primary antibody. Hamster samples were immunoblotted with mAb 3F4 (1:10,000 dilution, Chemicon, Temecula, CA) which has a middle region epitope. Elk samples were immunoblotted with mAb L42 (1:200, R-Biopharm, Marshall, MI) which also has a middle region epitope. Pellets were washed with DPBS, incubated with secondary antibody (horseradish-peroxidase conjugated antimouse IgG, 1:2000, Pierce, Rockford, IL), and washed with DPBS. Low speed centrifugation (100g for 5 s) was used to remove all reagents, antibody solutions, and DPBS washes from soil pellets. Pellets were loaded into 96well PVDF plates (Pall Corp., Ann Arbor, MI), developed with brain homogenate controls using Pierce Supersignal West Femto maximum-sensitivity substrate, according to the manufacturer’s instructions (Pierce, Rockford, IL), and imaged on a Kodak 2000R imaging station (Kodak, Rochester, NY). All soil pellets were run in triplicate. Pellets were diluted if necessary when loaded into the 96-well plate to have the resulting signal within the linear range of the control dilution standard. Because multiple centrifugations and decantations are required, this method may select for certain soil particles. However, this selection would be constant for all samples, and mass recoveries indicate that the procedure does not remove large populations of PrP (13).

Quantification of PrP. For each direct detection assay experiment, control serial dilutions of brain homogenate using the same tissue, antibody, and PK treatment were performed in triplicate. The relative abundance of PrP in samples was determined by a linear regression of the serial dilutions as described previously (13). Because the absolute amount of PrP in any given tissue homogenate cannot be measured at this time, sample quantities are reported in µg of brain equivalents (BE), where 1 µg BE equals 1 µg starting brain tissue (hamster or elk). A 1.0% brain homogenate dose ) 10 µg brain equivalents/µL. See Figure S1 for a typical linear regression and the Supplemental Methods Discussion for further details on PrP quantification.

Results Kinetic Studies of PrP Adsorption. Two starting brain homogenate concentrations (doses) were used, 0.5% and 2.5%, for kinetic studies of PrP adsorption to sand, sandy loam, and silty clay loam soil. We were unable to quantify CWD-elk and uninfected-hamster (UN) adsorption in µg brain equivalents (BE) due to inconsistencies in the serial dilutions of the 96-well immunoassay (data not shown). This may have been due to the use of a different antibody for the elk samples (L42 vs 3F4) or due to a lower level of PrP in both the uninfected and elk samples. However, soil pellets from CWD and UN sorption experiments were normalized as a percentage of a constant brain homogenate control run concurrently (six control replicates per run, Figure 1A). Samples could therefore be compared to establish kinetic and isothermal trends. Adsorption of DY TME PrP to fine quartz sand reached a maximum of approximately 0.5 µg BE/mg sand after 1 week and remained relatively constant through 40 d but did increase to 1.3 µg BE/mg at the 0.5% dose (Figure 1B and C). Maximum adsorption at the 2.5% dose was 2.5 µg BE/mg. These values are 60% and 50% less, respectively, than the maximum adsorption previously reported for HY PrP (13). Total aqueous DY PrP decreased over time through 60 d (Figure S1), mirroring behavior for HY PrP as reported previously (13). Adsorption of CWD-elk PrP to sand increased continually through 60 d, with the sorbed fraction determined after a 1 h equilibration time representing only 4% of the maximum (Figure 1B and D). Uninfected hamster PrPc adsorption to sand was maximal at 40 d and decreased by 50% at 60 d (Figure 1D). PrPc adsorption after an equilibration period of 1 h was 9% of the maximum. Adsorption of CWD-elk PrP to sandy loam increased continually through 50 d, with a maximum normalized intensity of 6 (Figure 1E). There was no difference in adsorption between the doses at 20 d. CWD results were similar to previously reported HY TME results through 20 d, but CWD adsorption was 3 times higher at 50 d. CWD-elk PrP adsorption to silty clay loam appeared to reach a maximum after 1 week, with a maximum normalized intensity of approximately 20 (Figure 1F). CWD results for silty clay loam closely mirrored previous HY TME results (13). Isothermal Studies of PrP Adsorption. Isotherm experiments were conducted to determine sorption behavior as a function of concentration. Because maximum PrP adsorption in brain homogenate does not occur before 1 week for most soils (13), we used 1 week as a standard equilibration time. Mass ratios of total BE to soil were varied from 1:0.05 to 1:20. Maximum adsorption of HY TME PrP occurred at an intermediate aqueous concentration for sand and both whole soils (Figure 2B, D, and E). For sand, an approximately linear increase in Cs occurred as Cw increased to 10 µg BE/µL (Figure 3A). Thereafter, as Cw increased, Cs gradually decreased through a Cw of 50 µg BE/µL. HY PrP adsorption to sandy loam soil had a maximum Cs at 5 µg BE/µL and similarly VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Kinetic studies of DY TME, CWD-elk, and uninfected PrP adsorption. (A) 2-fold serial dilution of DY TME brain homogenate and control blots for CWD-elk and uninfected (hamster) brain homogenates. DY and uninfected detected with mAb 3F4, CWD detected with mAb L42. (B) Fine quartz sand results. Brain homogenate (0.5 or 2.5% initial dose with 1.0% ) 10 µg brain equivalents/µL) was incubated with 10 mg of sand for up to 60 d at pH 7, 22 °C. Blots are representative of three replicates. (C) Quantified results for HY TME and DY TME PrPSc adsorption to sand. (D) Quantified elk-CWD PrP and uninfected hamster (PrPc) adsorption to sand. (E) Quantified CWD and HY PrP adsorption to sandy loam soil. Blot images are not shown. (F) Quantified CWD and HY PrP adsorption to silty clay loam soil. Blot images are not shown. All HY TME data are from ref 13 and shown for comparison. Error bars show ( the standard error. Note: panels D-F show normalized intensities while panel C shows Cs in µg BE/ mg sand. decreased at higher Cw (Figure 3C). At Cw of 50 µg BE/µL, Cs was near zero. HY PrP adsorption to silty clay loam had an intermediate maximum between 5 and 15 µg BE/µL (Figure 3D). An HY-sand isotherm experiment conducted with a 1 h equilibration time did not have an intermediate maximum (Figure S3). DY TME PrP adsorption was consistently lower than HY PrP for all sorbents evaluated (Figure 2A, C, and D). DY isotherm shapes were similar to those of HY (Figure 3A, C, and D). DY PrP adsorption was on average 60%, 65%, and 90% lower than HY for sand, sandy loam, and silty clay loam soils, respectively. In contrast to HY and DY PrP, CWD-elk PrP adsorption was maximal at the lowest concentration tested and decreased gradually as the aqueous concentration increased (Figures 2C and 3B). However, CWD-elk PrP did exhibit intermediate maximum behavior with a 1 h equilibration 5244

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(Figure S4). Uninfected hamster brain homogenate (PrPc) did not exhibit an intermediate maximum (Figures 2C and 3B). Adsorption was constant at low and intermediate aqueous concentrations and decreased somewhat at higher concentrations. Isotherms were generated using PK-digested HY TME brain homogenate. The N-domain of PrPSc is removed by proteinase K digestion, which also completely degrades PrPc (30). PK digested PrPSc adsorption to sand was on average 8-fold greater than PrP in undigested homogenate (Figure 3A). Adsorption of digested PrPSc to sandy loam was on average 2-fold greater than undigested PrP (Figure 3C). In contrast, digested PrPSc adsorption to silty clay loam soil was on average 50% less than undigested PrP (Figure 3D). Isotherm shapes for PK-digested samples were similar to undigested results, with intermediate maxima at similar aqueous concentrations.

FIGURE 2. Representative immunoblots from isothermal studies of PrP adsorption. (A) Representative immunoblot of HY TME brain homogenate serial dilutions. (B) Sand isotherm results. HY TME, DY TME, and PK-digested HY TME brain homogenates were incubated with 10 mg fine quartz sand. (C) CWD-elk and uninfected (hamster) PrP adsorption to sand. (D) Sandy loam soil results. Pellet was 3 mg of soil. HY PK+ pellet was diluted 1:3. (E) Silty clay loam results. Pellet was 1 mg of soil. HY PK+ and HY PK- pellets were diluted 1:25; DY pellets were diluted 1:15. All panels detected with mAb 3F4 except Line 1, panel C, detected with mAb L42. All isotherms had a 168 h (1 week) incubation time at pH 7, 22 °C.

Discussion PrP Adsorption Varies with Species and Strain. Recent evidence suggests rodent-adapted prions may not fully simulate the fate of natural prion disease in the environment (4, 30).Thus, it is important to confirm experimental results with a more environmentally relevant model, such as CWD or scrapie-infected tissue (4). The results presented here provide further evidence that rodent models may not recapitulate CWD. In the kinetic studies, HY PrP adsorption plateaued after 30 d, while CWD PrP adsorption continued to increase through at least 60 d for sand and sandy loam soil. CWD PrP does not degrade significantly through 30 d in the presence of in vivo brain proteases, while HY PrP is highly degraded after 30 d (30). These observations may explain the continued increase in CWD adsorption to sand and sandy loam soil through 60 d whereas HY reached a maximum around 30 d, corresponding to the near-complete degradation of HY PrP in the liquid fraction. Furthermore, in contrast to HY and DY PrP, adsorption of CWD PrP did not exhibit an intermediate maximum after a 1 week equilibration time in isothermal experiments (Figure 3B). However, a 1 h equilibration time did yield an inter-

mediate maximum (Figure S4). It is possible that CWD PrP adsorption peaked at a very low aqueous concentration (between 0 and 3 µg BE/µL) for the 1 week isotherm. The concentration of PrP in the infected elk brain may have been lower than PrP in the hamster brains, in which case CWD PrP adsorption may have peaked at a lower aqueous concentration due to a comparatively greater amount of competing proteins. The significance of the intermediate maximum is discussed in detail below. Strain differences in PrP adsorption were also observed. HY TME PrP consistently adsorbed in higher quantities than DY TME PrP in both the kinetic and isothermal studies. HY and DY are both hamster strains and therefore exist in similar brain homogenate matrices but differ in the three-dimensional structure of PrPSc (22-24, 26). It is possible that equal amounts of DY and HY PrP adsorb, but that the resulting conformation or orientation of bound DY PrP yields a decreased affinity for the primary antibody (mAb 3F4) compared with bound HY PrP. This would imply different adsorption mechanisms between the two strains. DY and HY PrP affinity for mAb 3F4 on a PVDF membrane is indistinguishable (data not shown). The aqueous concentration of DY PrP closely mirrored that of HY PrP during the kinetic study (Figure S2), so it is unlikely that the difference in adsorption is due to the availability of PrP in solution. Since the conformational differences between HY and DY PrPSc are not fully defined, it is difficult to explain the stronger adsorption affinity of HY PrP. Differences in PrPSc aggregation may be responsible. The observed difference in DY and HY adsorption was approximately equal for sand and sandy loam soil but much greater for silty clay loam soil. This larger difference could be due to the increased surface area of the soil or to a reduced affinity of DY to hydrophobic surfaces on the clay. DY PrPSc is known to be more soluble than HY PrPSc in the presence of detergents (22), which may indicate it is less hydrophobic. Fundamental information on PrPSc structure and strain variance is needed before differences in adsorption can be explained. Although no CWD strains have been identified, evidence suggests they may exist (31). Our data indicate that environmental pressures such as adsorption to soil could significantly alter the fate of prions based on strain properties, affecting mobility and susceptibility to degradation. Our results also caution that the fate of scrapie, CWD, and BSE in soil environments may be different and each requires separate investigation. Adsorption of N-Terminally Truncated PrPSc. A large population of PrP will enter and exist in the environment without the N-terminal domain (30). Because the N-terminal domain is known to be flexibly disordered and contain a high number of positively charged amino acids, it may play a significant role in electrostatic attraction to negatively charged mineral surfaces (20). Evidence suggests the Ndomain might play a role in sorption to clays (4). However, our results confirm previous reports that the N-domain is not needed for prion adsorption (4, 16). We found that N-terminally truncated PrPSc in a PK-digested brain homogenate adsorbs more readily to sand and sandy loam soil but less readily to silty clay loam soil compared with undigested (full-length) PrP. This result is in agreement with a previous observation that more full-length recPrP adsorbs to montmorillonite clay than N-terminally truncated recPrP (32). Increased adsorption of truncated PrPSc to sand and sandy soils has not been previously demonstrated, and the underlying reasons for the observed behavior are unclear. The N-domain of PrPSc is susceptible to proteases and therefore is likely on the exterior of the molecule (30). Removal of the N-domain may eliminate steric hindrances, allowing PrPSc to adsorb more readily to quartz surfaces. The overall decrease in prion protein mass and size may also alter adsorption. VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Quantified isothermal studies of PrP adsorption. Quantification of PrP adsorption data from immunoblots shown in Figure 2. Cs (solid fraction) versus starting Cw (aqueous fraction) (A) HY PK+, HY PK-, and DY PK- PrP adsorption to fine quartz sand. (B) CWD-elk and uninfected (hamster) PrP adsorption to sand reported as normalized intensities. HY results are from Panel A. (C) Adsorption of HY PK+, HY PK-, and DY PK- PrP to sandy loam soil. (D) Adsorption of HY PK+, HY PK-, and DY PK- PrP to silty clay loam. Error bars show ( the standard error. N g 3 for all points except 50 µg BE/µL points in A, B, and C and 100 µg BE/µL in D, where n ) 1. Finally, PK-digestion may modify PrPSc aggregation, yielding a conformation that promotes interaction with quartz surfaces but is less preferential for clay surfaces. Changes in the brain homogenate matrix due to PK-digestion may be partially responsible for differences in truncated and full-length PrPSc adsorption. A coomassie blue stain comparing the total protein before and after PK-digestion indicated a 60% decrease in protein content upon digestion, with a majority of the remaining peptides less than 14 kDa (Figure S5). Thus, PKdigested homogenate has fewer large polypeptides than undigested homogenate to compete for adsorption sites, possibly allowing more PrPSc to adsorb. In addition, PK-digestion may reduce constituents which favorably or unfavorably coat or modify soil surfaces. It is unclear how significant these factors are, hampering interpretation of the results. Nevertheless, our data provide evidence that the presence of the N-terminal enhances adsorption of PrPSc to clay but may hinder adsorption to sand. Conceptual Model of Prion Adsorption in a Competitive Matrix. Prions enter the environment in multiconstituent organic mixtures (e.g., tissue, feces, urine, saliva) and interact with complex soil environments in which constituents will degrade, adsorb and desorb, and reorient or change conformations once associated with a surface (4). We used infected brain homogenates as a more complex, relevant model for prion interactions with soil compared with previous studies using recPrP or purified PrPSc. We previously found that this competitive matrix slows and reduces PrP adsorption by several orders of magnitude (13). In the current study, we document an intermediate maximum sorption phenomenon in isothermal studies, where PrP adsorption increases linearly 5246

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FIGURE 4. Model for PrP adsorption to soil in a competitive matrix. Adapted from the Vroman effect (34). through an intermediate aqueous PrP concentration and then decreases at higher concentrations (Figure 4). Interestingly, isothermal studies of protein adsorption using blood plasma have found results similar to the ones presented here, with a maximum Cs at an intermediate plasma dilution (Cw). Dubbed the “Vroman effect”, this behavior has been observed with a variety of surfaces and proteins, although most studies have used fibrinogen, a protein present in large quantities in plasma (33, 34). At low homogenate concentrations, proteins are free to adsorb onto the unsaturated surface. At higher homogenate concentrations, proteins or other

constituents with higher surface affinities outcompete proteins with lower surface affinities. This is a possible explanation for the observed isothermal behavior of PrP (Figure 4). An alternate explanation is that PrP adsorption remains constant at higher aqueous concentrations but is obscured from antibody binding by subsequent adsorption of other molecules. However, the Vroman effect has been confirmed with nonimmunological methods (34). It is important to note that tissue homogenate is more complex than blood plasma, which has fewer constituents and is primarily composed of select proteins such as fibrinogen and albumin. Another component of the Vroman effect seen with fibrinogen and other plasma proteins is maximum adsorption at intermediate contact times, due to displacement over time by other less abundant proteins with higher surface affinities (33, 34). In contrast, prion adsorption does not decrease over time after reaching maximum adsorption (13). Prion adsorption likely differs from fibrinogen adsorption in two respects. First, while fibrinogen is a large constituent of plasma, prions are trace proteins in vivo (35). Prion concentrations are probably equal to or less than that of proteins with stronger surface affinities, and it is therefore unlikely that prions will initially overshoot the equilibrium maximum as fibrinogen does. Second, prion adsorption is largely irreversible whereas fibrinogen is readily displaced by other proteins. Once adsorbed, prions are not likely to be replaced by other constituents. PrPSc aggregation may be an important factor in adsorption. In contrast to infectious tissue isotherms, PrPc (not aggregated) in uninfected hamster brain homogenate did not exhibit a distinct intermediate maximum (Figure 3B). Furthermore, a recent study reported accelerated fibrillation of a prion peptide on a silicon surface compared with peptide fibrillation in solution (36), and aggregation of other proteins upon adsorption has also been shown (37, 38). The data presented in this paper show that the maximum Cs and corresponding intermediate Cw values can vary with soil as well as prion strain and species brain homogenate. Other matrices in which prions might enter soil environments (e.g., feces, urine, and saliva) are similarly competitive in nature but consist of different constituents and a lower concentration of PrPSc. It is likely that an intermediate maximum would also be characteristic of PrPSc adsorption in these matrices. However, the maximum Cs and equilibration time for these matrices could be different than when PrPSc is introduced in brain homogenate. Thus, the environmental fate of prions may vary greatly depending on the fluid or tissue in which it enters the environment. These effects could be a more significant determinant of CWD and scrapie transmission in the environment than the overall mass of PrPSc entering the environment. PrP adsorption to soil in other competitive matrices requires further investigation.

Acknowledgments We thank Ronald Shikiya and Michelle Kramer for technical assistance and Ken Clinkenbeard for the CWD-elk brain. This research was supported in part by the UNL Research Council and the National Center for Research Resources (P20 RR0115635-6 and C06 RR17417-01).

Supporting Information Available Table S1, Soil and Soil Mineral Characteristics; Table S2, Soil Mineralogy (Weight Percent); Supplemental Methods Discussion; Figure S1, Comparison of Typical Linear Regressions of 96-Well Control Dilutions vs Typical Western Blot Quantification; Figure S2, Total Aqueous DY TME PrP Quantified Over Time; Figure S3, HY 1 h Sand Isotherm; Figure S4, CWD 1 h Sand Isotherm; Figure S5, Total Protein Degradation Due to PK Digestion. This material is available free of charge via the Internet at http://pubs.acs.org.

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