Effects of solution chemistry and aging time on prion protein adsorption and replication of soil-bound prions

Abstract

Prion interactions with soil may play an important role in the transmission of chronic wasting disease (CWD) and scrapie. Prions are known to bind to a wide range of soil surfaces, but the effects of adsorption solution chemistry and long-term soil binding on prion fate and transmission risk are unknown. We investigated HY TME prion protein (PrP(Sc)) adsorption to soil minerals in aqueous solutions of phosphate buffered saline (PBS), sodium chloride, calcium chloride, and deionized water using western blotting. The replication efficiency of bound prions following adsorption in these solutions was also evaluated by protein misfolding cyclic amplification (PMCA). Aging studies investigated PrP(Sc) desorption and replication efficiency up to one year following adsorption in PBS or DI water. Results indicate that adsorption solution chemistry can affect subsequent prion replication or desorption ability, especially after incubation periods of 30 d or longer. Observed effects were minor over the short-term (7 d or less). Results of long-term aging experiments demonstrate that unbound prions or prions bound to a diverse range of soil surfaces can readily replicate after one year. Our results suggest that while prion-soil interactions can vary with solution chemistry, prions bound to soil could remain a risk for transmitting prion diseases after months in the environment.

Figure 1. Solution Chemistry Does Not Affect Short-Term HY TME PrP^Sc Adsorption to Bentonite Clay and Silicon Dioxide Powder.

(A): Representative Western blots of PrP^Sc adsorbed to bentonite clay or silicon dioxide powder (SiO₂) in DPBS, CalCl₂, or NaCl solutions for 1 h, 1 d, or 7 d at 22°C. ‘Control’ indicates 250 µg eq clarified HY TME brain control (DPBS for lane 1, H₂O for lanes 5 and 9). (B): Approximate PrP^Sc recovery in soil pellets (‘Bound’) and supernatants (‘Unbound’). Error bars represent ±1 standard error of the mean.

Figure 2. Effect of Adsorption Solution on Replication Efficiency of Soil Mineral-Bound HY TME.

(A–D): Representative Western blots of HY samples subjected or not subjected to PMCA, shown with a 2 µl 10% BH control. (A): HY brain tissue homogenized in DPBS or H₂O. (B): SiO₂ powder-bound HY (one round PMCA). (C): First round of bentonite clay-bound HY. (D): Second round of bentonite clay-bound HY. (E): Quantification of blots shown in (A–D). Amplified signal was calculated by normalizing sample intensities to HY DPBS BH controls subjected to PMCA concurrently. Error bars show ±1 standard error of the mean. *Denotes significant difference (p<0.01).

Figure 3. Effect of Adsorption Time and Adsorption Solution on Sand-Bound HY TME Replication.

Amplified PrP^Sc signals from one round of PMCA of HY TME sand samples. Amplified PrP^Sc signals were normalized to HY DPBS BH controls subjected to PMCA concurrently. Error bars show ±1 standard error of the mean. Representative Western blots shown in Figure S3.

Figure 4. Long-Term Aging of Unbound and Soil-Bound HY TME PrP^Sc.

(A–D): Representative Western blots (n = 3) of aged HY TME samples, shown with a 2.5 µl 10% BH control. Arrows indicate migration of 29 kDa and 20 kDa molecular weight markers. (A): 10% HY brain homogenate. (B): Bentonite clay. (C): Silicon dioxide powder. (D): Rinda silty clay loam soil (SCL Soil). (E–F): Quantification of blots shown in (A–D). Error bars show ±1 standard error of the mean. *indicates sample (BH H₂O 180 d) not available.

Figure 5. Soil-Bound HY TME Prions Remain Replication Competent after 1 Year Incubation.

Amplified PrP^Sc signals from one round of PMCA of HY TME unbound or bound to various soils. Amplified PrP^Sc signals were normalized to HY DPBS BH controls subjected to PMCA concurrently. Error bars show ±1 standard error of the mean. Representative Western blots shown in Figure S4.