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Prion protein is identified in human brains as “protease-resistant.” That is, it resists degradation by an enzyme that degrades proteins in general. But researchers have discovered an enzyme that can degrade the prion protein held responsible for mad cow disease and other transmissible spongiform encephalopathies (TSEs).

“If animal feed or food is contaminated with a prion, we can use this enzyme for disinfection and decontamination,” said Jason Shih, PhD, Professor of Biotechnology and Poultry Science at North Carolina State University in Raleigh.

Transmissible prions – believed to be the cause of bovine spongiform encephalopathy (BSE), as well as the human and sheep versions, called variant Creutzfeldt-Jakob disease in humans and scrapie in sheep – are highly resistant to degradation, Dr. Shih said.

The new research tested the effects of the bacterial enzyme keratinase on brain tissues from cows with BSE and sheep with scrapie. When the tissue was pretreated and in the presence of a detergent, the enzyme fully degraded the prion, rendering it undetectable.

“We reduced the prion to undetectable levels,” Dr. Shih said.

The research was published in the Journal of Infectious Diseases (2003;188:1782–1789).


The story started 20 years ago, when Dr. Shih, seeking to find better technology for animal waste management, developed a process that converts poultry manure to a gas, methane.

“During the process, I observed that all the feathers dissipated,” he said. “That meant some microorganism must have been breaking them down.”

Keratin, a versatile recalcitrant protein that protects the animal's body, makes up more than 90 percent of a feather's content, he said. This is true of all kinds of birds.

Working in the laboratory, Dr. Shih isolated a bacterium, Bacillus licheniformis strain PWD-1, which can break down feathers. “It turned out that the bacterium secretes keratinase, which is the enzyme that breaks down the feathers,” he said.

The researchers then purified the enzyme, isolated and sequenced its genes, and modified the protease for hyperproduction of the enzyme, he said. Using permutation technologies, they were able to produce it in mass quantities. Now the trick was to develop an application process, he said. There are three areas of interest: using the enzyme to convert feathers into a digestible protein to feed animals; using the enzyme as a feed additive to improve the digestibility of the material; and prion inactivation.

Dr. Shih found that adding keratinase to chicken feed increased both its digestibility and the efficiency. Chickens fed keratinase-enriched feed grew to optimal weight quicker, while eating less feed than normal. “It's the same combination of benefits associated with antibiotics, but appealing to people looking for safer feed substitutes,” he said.


Dr. Jason Shih: “The implication is that this enzyme can be used for prion inactivation, so if feed or hospital equipment is contaminated, we could use this enzyme for disinfection and decontamination.”


Dr. Shih next turned his attention to prion inactivation. Feather keratin has a protein structure similar to that of the prion, he said. “It's a rich, beta sheet structure, similar to the pathogenic unfolded isoform of prions.”

This same structure that makes feathers resistant to hydrolysis, he said, explains why prions generate resistance to the proteolytic enzyme.

With that in mind, the researchers brought the enzyme to the Netherlands, where the Centers for Animal Disease Control in Lelystad had an assay that could detect the disease-associated isoform of the prion.

Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, the researchers tested conditions needed to accomplish the full degradation of prion protein in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie.

They found that, in the presence of detergents, heat pretreatment with the enzyme at over 100°C allowed extensive enzymatic breakdown of the prion protein to a state where it was immunochemically undetectable.

“It turned out the enzyme broke down prions,” Dr. Shih said. “The implication is that this enzyme can be used for prion inactivation, so if feed or hospital equipment is contaminated, we could use this enzyme for disinfection and decontamination.”

Currently, if food or an animal is contaminated, the only way to disinfect it is by burning, which is dangerous, he said. A sodium hydroxyl wash can be used to disinfect equipment, but it is caustic and harsh to equipment.

“This enzyme gives us a gentler method for cleanup,” Dr. Shih said. “Fear of contamination and the difficulty of clean-up inhibits the performance of brain biopsies and postmortem examination – so this could have practical advantages.”

But whether one could decontaminate an infected animal is not yet known, Dr. Shih said. The next step, he said, is a two-year study, funded by the National Cattleman's Beef Association, to test the effectiveness of the enzyme on the treated BSE prions in mice.

“We've reduced the prion to undetectable levels in vitro,” he said. “The mouse study will show whether these undetectable levels of prion are non-infectious.”

In conjunction with BioResource International, a North Carolina State spin-off biotechnology company, the researchers will also test the effectiveness of keratinase in decontaminating equipment that processes animal by-products. Many scientists believe that mad cow disease is spread by healthy animals eating feed containing by-products from BSE-infected animals.

“Using keratinase to destroy prions on processing equipment could help to lower the risk of spreading BSE,” Dr. Shih said.


Other researchers were cautiously optimistic. James Mastrianni, MD, PhD, Assistant Professor of Neurology at the University of Chicago, said, “an enzyme that would degrade prions effectively and easily would be a real advance.”

“The approach sounds reasonable,” he said, “though we'll have to wait and see how it pans out in future studies.”

Patrick Bosque, MD, Assistant Professor in the Department of Neurology at the University of Colorado Health Sciences Center at Denver, agreed. “The research is interesting, but whether it is a breakthrough is too early to tell.”

Currently, “if you work on a patient with prion disease, you just throw away the tools,” he said. “If this protease system could be optimized, it could help us in terms of infection control – sterilizing hospital and farm equipment.”

“But as for treating the food directly, at least for now, I would have reservations,” Dr. Bosque said. “Decontaminating a steak in this manner would dissolve it.”

According to Dr. Mastrianni, there are two major focuses to current prion research: finding a sensitive and specific test for early detection of prion diseases in asymptomatic individuals and animals and developing an effective drug therapy.

“Because it's primarily a brain disease, detection in the blood or tissue is difficult,” he explained. “Once we have a test, though, it would be much easier to develop a drug to treat it.”


✓ Researchers have discovered an enzyme that can degrade the prion protein held responsible for mad cow disease and other transmissible spongiform encephalopathies.


For years, prions themselves have been one of medicine's biggest mysteries, researchers say. It wasn't until the latter part of the century that researchers homed in on prions as the cause of TSEs. Before their discovery, infectious diseases were thought to be transmitted only by viruses or other microorganisms that contains nucleic acid, allowing them to multiply in the infected host.

But prions are pure proteins. “We all have normal prion proteins,” explained James Mastrianni, MD, PhD, Assistant Professor of Neurology at the University of Chicago. “Infectious prion proteins are just an altered shape, a modified form of the ‘normal’ protein” that are no longer vulnerable to proteases.

Experiments in the 1980s by Stanley Prusiner, MD, of the University of California-San Francisco, showed prions not only caused TSEs, they also were a form of protein that occurs naturally in all mammalian cells.

Once prions infect the body, they cannot be destroyed. As they accumulate, the misshapen proteins somehow trigger neighbor proteins to behave similarly, eventually taking the place of normal proteins and destroying brain cells.

Giuseppe Legname, PhD, an Assistant Adjunct Professor of Neurology who works in the lab of Stanley Prusiner, MD, at the University of California-San Francisco, said, “It was an important discovery because we thought propagation of an infectious agent had to go through nucleic acid. This was a whole new way of thinking about infectious agents.”

About the same time as Dr. Prusiner made his startling discovery, an epidemic of bovine spongiform encephalopathy (BSE) began in the United Kingdom. No one is sure how the animals caught BSE, but scientists believe they became infected after eating feed that had been made from scrapie-infected animals.

It is believed that humans with mad cow disease, known scientifically as new variant Creutzfeldt-Jacob disease (vCJD), then became infected after eating cattle products contaminated with BSE-infected central nervous system tissue.

“There is really no difference between BSE and vCJD,” Dr. Mastrianni said. “It's the same process, the same infectious agent, the same mechanisms. In the United Kingdom, all the people who got it ate infected feed.”

In both cases, “the infectious protein converts the normal prion in the brain to the abnormal form, causing neurodegeneration and eventually cell death,” he said.

So how is scrapie converted to BSE, or BSE converted to human disease?

“That's the million dollar question,” Dr. Legname said. “We don't know. There is a so-called species barrier where you cannot easily infect one species with another species' prion. The problem is that with BSE, this barrier has been broken.”

Even while continuing to unravel the mysteries of TSEs, researchers are working to develop treatments to control the disease. One approach that is now being tested in humans is the anti-malaria drug quinacrine, he said. Quinacrine had inhibited the spread of prions in a mouse neuroblastoma cell line infected with scrapie prions (Proc Nat Acad Sci USA 2001;98:9836–9841).

“But it is still early,” Dr. Legname said. In the meantime, patients should avoid any food that could be contaminated.

Creation of a vaccine has also been difficult, because “we all make this protein,” Dr. Mastrianni said. “Passive immunization would work theoretically, but we have to find a passive antibody specific for the abnormal form of the protein. And that is proving difficult.”


• Langeveld JPM, Wang J, Van de Wiel DFM, Enzymatic degradation of prion protein from infected cattle to sheep. J Infect Dis 2003;188:1782–1789.