Presence and Seeding Activity of Pathological Prion Protein (PrPTSE) in Skeletal Muscles of White-Tailed Deer Infected with Chronic Wasting Disease

Chronic wasting disease (CWD) is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE), or prion disease, occurring in cervids such as white tailed-deer (WTD), mule deer or elk in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report for the first time a direct biochemical demonstration of pathological prion protein PrPTSE and of PrPTSE-associated seeding activity, the static and dynamic biochemical markers for biological prion infectivity, respectively, in skeletal muscles of CWD-infected cervids, i. e. WTD for which no clinical signs of CWD had been recognized. The presence of PrPTSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrPTSE was revealed by protein misfolding cyclic amplification (PMCA). Semi-quantitative Western blotting indicated that the concentration of PrPTSE in skeletal muscles of CWD-infected WTD was approximately 2000-10000 -fold lower than in brain tissue. Tissue-blot-analyses revealed that PrPTSE was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrPTSE in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans

Quantitative Detection and Biological Propagation of Scrapie Seeding Activity In Vitro Facilitate Use of Prions as Model Pathogens for Disinfection

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants

Disease Transmission by Misfolded Prion-Protein Isoforms, Prion-Like Amyloids, Functional Amyloids and the Central Dogma

In 1982, the term “prions” (proteinaceous infectious particles) was coined to specify a new principle of infection. A misfolded isoform of a cellular protein has been described as the causative agent of a fatal neurodegenerative disease. At the beginning of prion research scientists assumed that the infectious agent causing transmissible spongiform encephalopathy (TSE) was a virus, but some unconventional properties of these pathogens were difficult to bring in line with the prevailing viral model. The discovery that prions (obviously devoid of any coding nucleic acid) can store and transmit information similarly to DNA was initially even denoted as being “heretical” but is nowadays mainly accepted by the scientific community. This review describes, from a historical point of view, how the “protein-only hypothesis” expands the Central Dogma. Definition of both, the prion principle and the Central Dogma, have been essential steps to understand information storage and transfer within and among cells and organisms. Furthermore, the current understanding of the infectivity of prion-proteins after misfolding is summarized succinctly. Finally, prion-like amyloids and functional amyloids, as found in yeast and bacteria, will be discussed

Chronic wasting disease: Fingerprinting the culprit in risk assessments

Transmissible spongiform encephalopathies (prion diseases) in animals may be associated with a zoonotic risk potential for humans as shown by the occurrence of variant Creutzfeldt-Jakob disease in the wake of the bovine spongiform encephalopathy epidemic. Thus, the increasing exposure of humans in North America to cervid prions of chronic wasting disease (CWD) in elk and deer has prompted comprehensive risk assessments. The susceptibility of humans to CWD infections is currently under investigation in different studies using macaques as primate models. The necessity for such studies was recently reinforced when disease-associated prion protein and its seeding activity were detected in muscles of clinically inconspicuous CWD-infected white-tailed deer (WTD). Increasing evidence points to the existence of different CWD strains, and CWD prions may also change or newly emerge over time. Therefore, CWD isolates examined in macaques should be characterized as precisely as possible for their molecular identity. On this basis other CWD field samples collected in the past, present or future could be systematically compared with macaque-tested inocula in order to assess whether they are covered by the ongoing risk assessments in primates. CWD typing by Fourier transform-infrared spectroscopy of pathological prion protein may provide a method of choice for this purpose

Towards further reduction and replacement of animal bioassays in prion research by cell and protein misfolding cyclic amplification assays

Laboratory animals have long since been used extensively in bioassays for prions in order to quantify, usually in terms of median infective doses [ID50], how infectious these pathogens are in vivo. The identification of aberrant prion protein as the main component and self-replicating principle of prions has given rise to alternative approaches for prion titration. Such approaches often use protein misfolding cyclic amplification (PMCA) for the cell-free biochemical measurement of prion-associated seeding activity, or cell assays for the titration of in vitro infectivity. However, median seeding and cell culture infective doses (SD50 and CCID50, respectively) of prions are neither formally congruent nor definitely representative for ID50 titres in animals and can be therefore only tentatively translated into the latter. This may potentially impede the acceptance and use of alternative methods to animal bioassays in prion research. Thus, we suggest performing PMCA and cell assays jointly, and to check whether these profoundly different test principles deliver consistent results in order to strengthen the reliability and credibility of prion ID50 assessments by in vitro methods. With regard to this rationale, we describe three pairs of PMCA and glial cell assays for different hamster-adapted prion agents (the frequently used 263K scrapie strain, and 22A-H scrapie and BSE-H). In addition, we report on the adaptation of quantitative PMCA to human variant Creutzfeldt-Jakob disease (vCJD) prions on steel wires for prion disinfection studies. Our rationale and methodology can be systematically extended to other types of prions and used to further reduce or replace prion bioassays in rodents

The MalF P2 Loop of the ATP-Binding Cassette Transporter MalFGK2 from Escherichia coli and Salmonella enterica Serovar Typhimurium Interacts with Maltose Binding Protein (MalE) throughout the Catalytic Cycle ▿

We have investigated the interaction of the uncommonly large periplasmic P2 loop of the MalF subunit of the maltose ATP-binding cassette transporter (MalFGK2) from Escherichia coli and Salmonella enterica serovar Typhimurium with maltose binding protein (MalE) by site-specific chemical cross-linking in the assembled transport complex. We focused on possible distance changes between two pairs of residues of the P2 loop and MalE during the transport cycle. The distance between MalF(S205C) and MalE(T80C) (∼5 Å) remained unchanged under all conditions tested. Cross-linking did not affect the ATPase activity of the complex. The distance between MalF(T177C) and MalE(T31C) changed from ∼10 Å to ∼5 Å upon binding of ATP (or maltose, with a less pronounced result) and was reset to ∼10 Å after hydrolysis of one ATP. A cross-link (∼25 Å) between MalF(S205C) and MalE(T31C) was observed only when the transporter resided in a transition state-like conformation, as was the case after vanadate trapping or in a binding protein-independent mutant, both of which are characterized by tight binding of unliganded MalE to the transporter. Thus, we propose that the observed cross-link is indicative of catalytic intermediates of the transporter. Together, our results strengthen the notion that the MalF P2 loop plays an important role in intersubunit communication. In particular, this loop is involved in keeping MalE in close contact with the transporter. The data are discussed with respect to a crystal structure and current transport models

Reversible off and on switching of prion infectivity via removing and reinstalling prion sialylation

The innate immune system provides the first line of defense against pathogens. To recognize pathogens, this system detects a number of molecular features that discriminate pathogens from host cells, including terminal sialylation of cell surface glycans. Mammalian cell surfaces, but generally not microbial cell surfaces, have sialylated glycans. Prions or PrPSc are proteinaceous pathogens that lack coding nucleic acids but do possess sialylated glycans. We proposed that sialylation of PrPSc is essential for evading innate immunity and infecting a host. In this study, the sialylation status of PrPSc was reduced by replicating PrPSc in serial Protein Misfolding Cyclic Amplification using sialidase-treated PrPC substrate and then restored to original levels by replication using non-treated substrate. Upon intracerebral administration, all animals that received PrPSc with original or restored sialylation levels were infected, whereas none of the animals that received PrPSc with reduced sialylation were infected. Moreover, brains and spleens of animals from the latter group were completely cleared of prions. The current work established that the ability of prions to infect the host via intracerebral administration depends on PrPSc sialylation status. Remarkably, PrPSc infectivity could be switched off and on in a reversible manner by first removing and then restoring PrPSc sialylation

Chronic wasting disease

Transmissible spongiform encephalopathies (prion diseases) in animals may be associated with a zoonotic risk potential for humans as shown by the occurrence of variant Creutzfeldt-Jakob disease in the wake of the bovine spongiform encephalopathy epidemic. Thus, the increasing exposure of humans in North America to cervid prions of chronic wasting disease (CWD) in elk and deer has prompted comprehensive risk assessments. The susceptibility of humans to CWD infections is currently under investigation in different studies using macaques as primate models. The necessity for such studies was recently reinforced when disease-associated prion protein and its seeding activity were detected in muscles of clinically inconspicuous CWD-infected white-tailed deer (WTD). Increasing evidence points to the existence of different CWD strains and CWD prions may also change or newly emerge over time. Therefore, CWD isolates examined in macaques should be characterized as precisely as possible for their molecular identity. On this basis other CWD field samples collected in the past, present or future could be systematically compared with macaque-tested inocula in order to assess whether they are covered by the ongoing risk assessments in primates. CWD typing by Fourier transform-infrared spectroscopy of pathological prion protein may provide a method of choice for this purpose