MicrobiologyBytes

All scientific papers are important, but some are more important than others. Aside from its scientific importance, this paper is particularly important to me in purely personal terms. It comes from my own department. Ben Maddison was a PhD student in my laboratory many years ago and now heads up his own research group within the department. It’s also one of the final papers to come from Gary Whitelam, my former head of department, who died tragically last year. And as if all that wasn’t enough, as the UK starts to forget about how close we came to disaster with BSE, we’re still not completely sure that it’s all over.

Using the cutting-edge research technique of serial protein misfolding cyclic amplification (sPMCA), my colleagues show that prions are secreted in the milk from scrapie-exposed sheep. The sPMCA method involves incubating a small amount of abnormal prion with an excess of normal prion protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into misfolded prion.

Since scrapie is not transmissible to humans, these findings do not indicate the likely introduction of zoonotic prions from sheep into the human food chain. Nevertheless, the data do indicate caution in the risk assessment associated with such foods. Although it is unknown if analogous shedding of prions into milk occurs with bovine BSE, evidence from previous epidemiological and bioassay studies suggests that such a scenario seems unlikely to cause clinical disease. However, the present report strongly suggests that given the importance of cow’s milk in the human diet the potential presence of low levels of prions within milk warrants further investigation. Analyzing milk samples by sPMCA offers a methodology with clear potential for the identification of clinically sick animals and those with preclinical/subclinical prion disease. Such a non-invasive, live animal assay has the potential to contribute to the epidemiological study, management and control of prion diseases within farmed animals.

Prions are secreted in milk from clinically normal scrapie-exposed sheep. J Virol. Jun 3 2009. doi:10.1128/JVI.00051-09
The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically-affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset, irrespective of the immunohistochemical detection of protease-resistant PrP(Sc) within lymphoreticular and CNS tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a non-invasive methodology to detect scrapie during preclinical/subclinical disease.

Related:

• Prions in Milk
• The Origin of BSE
• What the heck are prions for?
• Fears over new wave of vCJD deaths

Stanley Prusiner was awarded the first ever SGM Prize Medal (to a microbiologist of international standing whose work has had a far-reaching impact beyond microbiology) at the SGM Spring meeting at Harrogate on 1st April 2009. MicrobiologyBytes was there and this is a summary of his Prize lecture.

Prions are infectious proteins which multiply by binding to a host cell protein and converting it into insolubile fibrils (“amyloid“). Prions are associated with infectious, inherited and sporadic diseases – a feature unique to these entities. Tikvah Alper was the first person to identify prions in the 1960s, but when Prusiner started working on them in 1974, at first he didn’t believe the protein-only hypothesis. After eight years of failing to be able to identify any nucleic acid associated with them, in 1982 he changed his mind and invented the name prion (“pree-on”).

In prion diseases, the cellular form of the protein, PrP^c, is converted into a disease-associated form, PrP^Sc. If prions really are infectious proteins, PrP^Sc produced in bacteria should be able to cause disease – and it does. It is also possible to produce synthetic amyloids with different biological properties – essentially strains of the protein.

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Quinacrine cures cultured cells of prions. In mice, the drug increases survival time of infected animals by up to 20%, but recently concluded clinical trials in humans have shown little effect. Prusiner’s group have found that quinacrine does work in stationary phase cells – such as those in the brain. Future trials of anti-prion (or amyloid) drugs need to be carried out in stationary cells. The latest assay uses genetically-modified mice which express luciferase when glial cells are disturbed. The resulting luminescence can be detected in the brains of live mice, and signs of disease can be recorded even before any neurological symptoms appear. This is up to eight times faster than waiting for the mice to die and examining their brains, and only requires one tenth of the animals. Prusiner hopes to use this approach to study Alzheimer’s and Parkinson’s disease, which also involve brain injury and amyloid deposits.

Stanley Prusiner’s take home message to all the students present was: it’s important to be lucky! But as Robin Weiss, SGM President, pointed out, Pasteur said: Fortune favours the prepared mind!

Related:

• Alzheimer’s Disease and Prions
• The Origin of BSE
• What the heck are prions for?
• Drugs for treatment of prion infections
• What drove the cow mad? Lessons from a fish

For over twenty years scientists have known that a normal protein in the brain, PrP, or prion protein, can become harmful and cause deadly illnesses like Creutzfeldt-Jakob disease (CJD) in humans, and bovine spongiform encephalopathy (BSE) in cattle. What they could not explain is why large amounts of this normal protein are produced by our bodies in the first place. In a new study, researchers reveal that PrP indeed plays a beneficial role for the organism – PrP helps cells communicate with one another during embryonic development.

In prion diseases, what transforms the normal PrP protein into a life-threatening substance is the abnormal alteration of its chemical structure. Moreover, prions have the treacherous ability to replicate by imprinting their abnormal structure into healthy PrPs, thereby generating new pathogenic particles. While this conversion process explains how prions are disseminated, an abnormal function of the prion protein is considered to be one of the reasons for neuronal degeneration. However, the normal function of PrP has remained an unsolved mystery for many years. All previous experiments in genetically modified mice had failed to provide conclusive evidence, as these animals lacking PrP seemed perfectly healthy. The scientists were able to show that the lack of PrP can cause clear physiological abnormalities in a living animal by using the tiny zebrafish as a model.

When the researchers microinjected zebrafish eggs with morpholinos, DNA-like molecules that prevent the normal production of PrP, the treated zebrafish embryos were unable to develop normally and eventually died. The proteins in the fish embryos normally found at cell-to-cell contact sites disappeared, rendering these cells unable to communicate and carry out the differentiation program that shapes the major structures of the body, including the nervous system. PrP serves as a glue element, bringing cells together and keeping them in contact. When two neighboring cells make contact, they become able to exchange important signals that affect the function of a tissue in the body. Although this work does not offer an immediate cure for CJD or BSE, it widens our understanding of prion diseases and provides hope for effective treatments.

Regulation of embryonic cell adhesion by the prion protein. 2009 PLoS Biol 7(3): e1000055
Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Caþ2-independent homophilic cell adhesion and signaling; and (2) modulates Caþ2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

Related:

• The Origin of BSE
• A novel approach in the molecular differentiation of prion strains
• What the heck are prions for?
• Alzheimer’s Disease and Prions

Prion diseases are a closely related group of fatal neurodegenerative disorders affecting the central nervous system of humans and animals. They include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru in humans; bovine spongiform encephalopathy (BSE) in cattle; and scrapie in sheep. The identification of variant CJD (vCJD) in the U.K. in 1996 and the subsequent experimental confirmation that BSE in cattle and vCJD in humans are caused by the same prion strain has led to a variety of concerns relating to public health. Iatrogenic transmission of classical (sporadic) CJD by a contaminated neurosurgical instrument has been reported, and epidemiological evidence suggests a fraction of apparently sporadic CJD may be caused by unrecognized iatrogenic infection during general surgery. The unknown but potentially substantial prevalence of clinically silent infection with vCJD prions in populations exposed to dietary BSE prions, together with the much wider tissue distribution of infectivity in vCJD, highlights the concerns of risk of infection through contact with surgical instruments. Secondary vCJD arising from blood transfusion has now been documented, indicating significant prionemia in asymptomatic donors during the incubation period. This wide distribution of infectivity makes common surgical and endoscopic procedures, in addition to neurosurgery and eye surgery, a potential risk factor for iatrogenic transmission of vCJD. Further, it is established that tissue prions withstand many forms of sterilization techniques and that the metal-adsorbed agent is even more resistant to both thermal and chemical treatments.

Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces. PNAS USA February 9, 2009
Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental advance was the cell culture-based scrapie cell assay, allowing prion titres to be estimated more precisely and an order of magnitude faster than by animal bioassays. Here we describe a bioassay method that exploits the marked binding affinity of prions to steel surfaces. Using steel wires as a concentrating and sensitization tool and combining with an adapted scrapie cell endpoint assay we can achieve, for mouse prions, a sensitivity 100X higher than that achieved in standard mouse bioassays. The rapidity and sensitivity of this assay offers a major advance over small animal bioassay in many aspects of prion research. In addition, its specific application in assay of metal-bound prions allows evaluation of novel prion decontamination methods.

Related:

• What the heck are prions for?
• Prion infectivity found in fat tissues of mice
• Prions in Milk
• Novel mutation linked to mad cow disease
• A novel approach in the molecular differentiation of prion strains

A decade ago, a new variant form of Creutzfeldt-Jakob disease was identified. The emergence of this prion disease in humans was the consequence of the zoonotic transmission of bovine spongiform encephalopathy through dietary exposure. Since then, the control of human exposure to prions has become a priority, and a policy based on the exclusion of known infectious materials from the food chain has been implemented. Because all investigations carried out failed to reveal evidence of infectivity in milk from affected ruminants, this product has continuously been considered as safe. In this study, researchers demonstrate the presence of prions in colostrum and milk from sheep incubating natural scrapie and displaying apparently healthy mammary glands. This finding indicates that milk from small ruminants could contribute to the transmission of prion disease between animals. It also raises some concern with regard to the risk to humans associated with milk products from ovine and other dairy species.

Prions in Milk from Ewes Incubating Natural Scrapie. 2008 PLoS Pathog 4(12): e1000238
Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

Related:

• The Origin of BSE
• A novel approach in the molecular differentiation of prion strains
• Oral transmission of prions is enhanced by binding to soil
• What the heck are prions for?
• Alzheimer’s Disease and Prions

Researchers have found novel prion infectivity in white and brown fat tissues of mice. Prion diseases, also known as transmissible spongiform encephalopathies, are infectious progressive fatal neurodegenerative diseases which affect humans as well as wild and domestic animals. Distribution of prion infectivity in organs and tissues is important in understanding prion disease pathogenesis and designing strategies to prevent prion infection in animals and humans. Previous studies in animals including sheep, goats, cattle, deer, mink, hamsters and mice, have found prion infectivity mostly in nervous system tissues such as the brain and spinal cord. The tissues studied in a mouse model demonstrate a proof of principle that white and brown fat tissues are sites of prion agent deposition and therefore may play a previously unrecognized role in prion infectivity and transmission of prion disease. The authors state clearly that it will be important to extend their studies to prion-infected large animals, such as cattle, sheep, deer, and elk where they may be potential sources of contamination of human and domestic animal food chains. Results of the current and future studies may merit additional consideration of steps to eliminate from the food chain any fat from ruminants suspected of exposure to or infection with prions.

Detection of Prion Infectivity in Fat Tissues of Scrapie-Infected Mice. 2008 PLoS Pathog 4 (12): e1000232
Distribution of prion infectivity in organs and tissues is important in understanding prion disease pathogenesis and designing strategies to prevent prion infection in animals and humans. Transmission of prion disease from cattle to humans resulted in banning human consumption of ruminant nervous system and certain other tissues. In the present study, we surveyed tissue distribution of prion infectivity in mice with prion disease. We show for the first time detection of infectivity in white and brown fat. Since high amounts of ruminant fat are consumed by humans and also incorporated into animal feed, fat-containing tissues may pose a previously unappreciated hazard for spread of prion infection.

Related:

• MicrobiologyBytes: Prions
• Prion Diseases
• How Now Mad Cow

The mother of a Spanish man who died from the human form of mad cow disease has also died from the illness. The mother, in her early 60s, died in August 2008. Her son, 41, died in February 2008. Three of these cases in Spain were in the same northern province, Leon. Researchers will try to determine whether the mother and son shared a genetic background making them more susceptible to contracting the illness.
The first confirmed death from mad cow disease in Spain was in 2005, when a young woman died near Madrid. In addition to the cases in Spain, as of June 2008 the following numbers of cases of human mad cow disease had been reported: 167 in the United Kingdom; 23 in France; 4 in Ireland; 3 in the United States; 2 each in Netherlands and Portugal; and one each in Canada, Italy, Japan and Saudi Arabia.

CNN

Related:

• Prion Diseases
• How Now Mad Cow
• Prions

Researchers from the United States Department of Agriculture have identified a novel mutation in the bovine prion protein gene in a cow confirmed with atypical Bovine Spongiform Encephalopathy (BSE). This is the first report of a confirmed case of BSE (aka mad cow disease) with a potential pathogenic mutation within the bovine Prnp gene. BSE, a transmissible spongiform encephalopathy (TSE) or prion disease of cattle, was first discovered in the United Kingdom in 1986. BSE is considered to be the cause of a human prion disease known as variant Creutzfeldt-Jakob Disease (vCJD). Veterinary scientists have now identified a novel mutation, E211K, in the bovine Prnp gene. This mutation is identical to the E200K pathogenic mutation in the human Prnp, which has been described as the most common cause of genetic CJD. The study supports the view that all three etiological forms of TSEs in humans are also present in cattle: infectious, sporadic, and genetic. It further supports the hypothesis that the BSE epidemic may have originated from a genetic case of cattle BSE. Cattle with similar mutations can be expected in cattle herds world-wide and could be the source of new BSE outbreaks. It is therefore critical to continue world-wide surveillance for typical and atypical BSE cases including sequencing of the Prnp gene. A newly developed assay system for detecting the E211K mutation has been developed for this purpose. Finally, in order to protect humans it is essential to continue to exclude Specified Risk Materials from the food chain and to maintain the ruminant feed ban.

BSE Case Associated with Prion Protein Gene Mutation. PLoS Pathog 4(9): e1000156
Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle. We hypothesize that the bovine Prnp E211K mutation most likely has caused BSE in the approximately 10-year-old cow carrying the E221K mutation.

Related:

• A novel approach in the molecular differentiation of prion strains
• Genes contributing to prion pathogenesis
• What the heck are prions for?
• Drugs for treatment of prion infections

The origin of the transmissible agent involved in the foodborne epidemic of bovine spongiform encephalopathy (BSE) remains a mystery. It has recently been proposed that this could have been the result of the recycling of an atypical, more probably sporadic, form of BSE (called bovine amyloidotic spongiform encephalopathy, or L-type BSE) in an intermediate host, such as sheep. A team from the French Food Safety Agency has identified a prion protein characteristic that is unique to some natural but unusual sheep scrapie cases. This finding may provide a novel method by which to study prion diversity and their possible changes during cross-species transmission. Mystery still surrounds the origin of the transmissible agent involved in the food-borne epidemic of bovine spongiform encephalopathy (BSE). Classical BSE, more commonly known as mad cow disease, is a known cause of a variant form of the incurable, degenerative neurological disorder Creutzfeldt-Jakob disease in humans. It has recently been proposed that this could have been the result of the recycling of an atypical, more probably sporadic form of BSE (called bovine amyloidotic spongiform encephalopathy, or L-type BSE) in an intermediate host, such as sheep. The team analyzed the molecular features of the disease-associated protease-resistant prion protein (PrPres) to determine any differences which might discriminate between scrapie and BSE cases. The researchers sampled PrPres from the brains of transgenic mice overexpressing the ovine prion protein after experimental infection with prions from bovine classical BSE, L-type BSE, and ovine scrapie. Scrapie cases were found to include rare “CH1641-Like” isolates, which share some PrPres molecular features with classical BSE and L-type BSE. The molecular features of the prion protein in the “CH1641-like” sheep scrapie cases more closely resemble those found in L-type BSE compared to classical BSE. However, from a series of four “CH1641-like” scrapie cases, the researchers found a pathological C-terminal prion protein product that was undetectable from both L-type and classical BSE transmitted to such mice, clearly suggesting that such scrapie isolates are not linked to these BSE forms. Further studies to confirm this discriminating factor are needed in sheep, especially from sheep experimentally infected with L-type BSE, which were not available for this study. These findings add a novel approach for the discrimination of prions that may help to understand their possible changes during cross-species transmissions.

A C-Terminal Protease-Resistant Prion Fragment Distinguishes Ovine “CH1641-Like” Scrapie from Bovine Classical and L-Type BSE in Ovine Transgenic Mice. 2008 PLoS Pathog 4(8): e1000137
The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.

Related:

• New forms of bovine spongiform encephalopathy
• Four separate types of Creutzfeldt-Jakob disease?
• Genes contributing to prion pathogenesis
• What the heck are prions for?