Prion protein interaction with soil humic substances: environmental implications

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment

Meat and bone meal as fertilizer-Nitrogen dynamics

The objective of this investigation was to evaluate nitrogen mineralization from meat and bone meals (MBM) subjected to different sanitization treatments (0.1M or 0.5M KOH in autoclave at 160 C for 30 min) according with UE Directives on TSE sanitization of residual animal by-products. Their overall nutritional efficiency was also tested cultivating maize (Zea mais) in pot experiments. The two treatments were compared with untreated MBM in leaching columns filled with sand or a 1:1 w/w mixture of soil and sand. Leached NH4+ from sand columns was similar (600 i'g N equivalent to 5% of total added N) for both treatments and untreated MBM, whereas it was negligible from soil during whole time course incubation. Nitrate-N leaching from sand columns was very low until day 40 and increased linearly thereafter for all treatments reaching values below 800 i'g N at day 100 (7-10% of added N). In the soil columns leached N-NO3- was already remarkably high from day 15 to 40 reaching cumulative values from 7 to 11 mg N (12, 30 and 49% of added N for untreated, 0.1M and 0.5M KOH treated MBM respectively)

Meat and bone meal as fertilizer-Nitrogen dynamics

The objective of this investigation was to evaluate nitrogen mineralization from meat and bone meals (MBM) subjected to different sanitization treatments (0.1M or 0.5M KOH in autoclave at 160 C for 30 min) according with UE Directives on TSE sanitization of residual animal by-products. Their overall nutritional efficiency was also tested cultivating maize (Zea mais) in pot experiments. The two treatments were compared with untreated MBM in leaching columns filled with sand or a 1:1 w/w mixture of soil and sand. Leached NH4+ from sand columns was similar (600 i'g N equivalent to 5% of total added N) for both treatments and untreated MBM, whereas it was negligible from soil during whole time course incubation. Nitrate-N leaching from sand columns was very low until day 40 and increased linearly thereafter for all treatments reaching values below 800 i'g N at day 100 (7-10% of added N). In the soil columns leached N-NO3- was already remarkably high from day 15 to 40 reaching cumulative values from 7 to 11 mg N (12, 30 and 49% of added N for untreated, 0.1M and 0.5M KOH treated MBM respectively)

Transformation of metamitron in a sandy clay loam soil

The degradation of metamitron, a sugar beet herbicide, in soil was studied in laboratory conditions and in field lysimeters under different irrigation water volumes. An identification of degradation compounds was also attempted. Laboratory degradation followed a first-order kinetics with a half-life of 18.2 days while lysimeter dissipation was consistently rapid with a half-life ranging from 4.4 to 8.0 days under the different irrigation conditions. A degradation compound appeared as early as the first few days in both laboratory and lysimeter samples and was identified as 4-(dimethylimino)-3-methyl-6-phenyl-1,2,4-triazin-5(4H)-one by GC/MS and NMR spectra. (© Inra/Elsevier, Paris.)Filière de transformation du métamitron dans un sol argilo-sableux. La dégradation dans le sol du métamitron, un herbicide de la betterave à sucre, a été étudiée en conditions de laboratoire et, sur le terrain, au moyen de lysimètres, sous différents volumes d'eau d'irrigation. On a également tenté d'identifier les composés de dégradation. La dégradation en laboratoire a suivi une cinétique de tout premier ordre, avec une demi-vie 18,2 j, tandis que la dissipation observée avec les lysimètres a été constamment rapide, la durée de demi-vie s'échelonnant entre 4.4 et 8 j suivant les différentes conditions d'irrigation. Un composant de dégradation est apparu dès les premiers jours tant dans les échantillons de laboratoire que dans ceux des lysimètres, et a, par le biais des analyses spectrales GC/MS et NMR, été identifié comme étant du 4-(diméthylimino)-3-méthyl-6-phényl-1,2,4-triazine-5(4H)-one. (© Inra/Elsevier, Paris.

Organic polyanions act as complexants of prion protein in soil

The persistence of prions, the causative agents of transmissible spongiform encephalopathies, in soil constitutes an environmental concern and substantial challenge. Experiments and theoretical modeling indicate that a particular class of natural polyanions diffused in soils and waters, generally referred to as humic substances (HSs), can participate in the adsorption of prions in soil in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks among humic acid molecules and exposed polar protein residues. Adsorption of HSs on clay surface strongly raises the adsorption capacity vs proteins suggesting new experiments in order to verify if this raises or lowers the prion infectivit

Organic polyanions act as complexants of prion protein in soil

5The persistence of prions, the causative agents of transmissible spongiform encephalopathies, in soil constitutes an environmental concern and substantial challenge. Experiments and theoretical modeling indicate that a particular class of natural polyanions diffused in soils and waters, generally referred to as humic substances (HSs), can participate in the adsorption of prions in soil in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks among humic acid molecules and exposed polar protein residues. Adsorption of HSs on clay surface strongly raises the adsorption capacity vs proteins suggesting new experiments in order to verify if this raises or lowers the prion infectivity.reservedmixedM. POLANO; C. ANSELMI; L. LEITA; A. NEGRO; DE NOBILI MPolano, Maurizio; C., Anselmi; L., Leita; A., Negro; DE NOBILI, Mari

Interactions of prion proteins with soil

Prions, are proteinaceous particles recognized as the agents of a class of neurodegenerative disorders, called transmissible spongiform encephalopathies (TSE), or prion diseases. Epidemiological data suggest that TSE-contaminated environments may serve as source of infectivity, but there is no information about adsorption of prions onto soil. We carried out experiments by mixing, healthy, or scrapieinfected hamster brains homogenates with three types of soil suspended in different buffers: (i) two saline buffers, i.e., phosphate buffer solution (PBS) and CaCl2 solution; (ii) a mix of nondenaturing detergents, i.e., Triton X-100 and sodium deoxycholate (DOC) solution; (iii) a non-ionic detergent, i.e., lauryl maltoside; (iv) two anionic detergents, i.e., Sarkosyl or sodium dodecyl sulphate (SDS); and (v) a chaotropic agent, i.e., urea. The unbound prion proteins were detected in the supernatants (after centrifugation of soil suspension) by Western blotting. Results clearly demonstrate that both the no infectious (PrP C ) and infectious (PrP Sc ) forms are adsorbed by all soils. Only 1% sodium dodecylsulphate (SDS) partially impeded the association of PrP C , but not that of PrP Sc with the sandy loam soil. Agents with different interacting properties towards hydrophilic and/or hydrophobic domains failed to extract PrP Sc from sediments of soil\u2013brain homogenate mixtures. The strong interaction of PrP Sc with soil favors the accumulation of prions in soils, especially if amended with prion-containing organic fertilizers and/or whenever TSE-affected animal carcasses, placenta, and excreta in general are buried or laid at the soil surface

Direct detection of soil-bound prions.

Scrapie and chronic wasting disease are contagious prion diseases affecting sheep and cervids, respectively. Studies have indicated that horizontal transmission is important in sustaining these epidemics, and that environmental contamination plays an important role in this. In the perspective of detecting prions in soil samples from the field by more direct methods than animal-based bioassays, we have developed a novel immuno-based approach that visualises in situ the major component (PrP(Sc)) of prions sorbed onto agricultural soil particles. Importantly, the protocol needs no extraction of the protein from soil. Using a cell-based assay of infectivity, we also report that samples of agricultural soil, or quartz sand, acquire prion infectivity after exposure to whole brain homogenates from prion-infected mice. Our data provide further support to the notion that prion-exposed soils retain infectivity, as recently determined in Syrian hamsters intracerebrally or orally challenged with contaminated soils. The cell approach of the potential infectivity of contaminated soil is faster and cheaper than classical animal-based bioassays. Although it suffers from limitations, e.g. it can currently test only a few mouse prion strains, the cell model can nevertheless be applied in its present form to understand how soil composition influences infectivity, and to test prion-inactivating procedures