Feasibility of infectious prion digestion using mild conditions and commercial subtilisin

Area of Science:

• Biochemistry
• Neuroscience
• Infectious Diseases

Background:

• Prion diseases are fatal neurodegenerative disorders caused by misfolded prion proteins (PrP(res)).
• Effective decontamination methods are crucial to prevent prion transmission, especially in healthcare settings.

Purpose of the Study:

• To investigate the efficacy of serine protease enzymes, specifically subtilisin 309 and subtilisin 309-v, in decontaminating prion-infected brain homogenates.
• To evaluate the potential of proteolytic digestion as a prion decontamination strategy.

Main Methods:

• Brain homogenates with high prion infectivity were digested using subtilisin 309 and subtilisin 309-v under mildly alkaline conditions.
• The Rocky Mountain Laboratory (RML) mouse-adapted scrapie model was used for bioassay to confirm elimination of PrP(res) infectivity.
• Comparison with established decontamination methods like autoclaving and hypochlorite treatment.

Main Results:

• One specific digestion condition (subtilisin 309, 138mAU/ml, 55°C, 14h, pH 7.9) showed statistically significant prion inactivation (P<0.001), with 52% mouse survival.
• Autoclaving (134°C) and hypochlorite (20,000 ppm) completely protected mice from prionosis.
• In vitro assays indicated that effective proteolytic decontamination requires high enzyme concentrations, pH >9.0, and elevated temperatures.

Conclusions:

• Proteolytic digestion using subtilisin enzymes under tested conditions is not a sufficiently effective method for prion decontamination.
• Achieving safe and efficacious prion inactivation via proteolysis necessitates conditions (high enzyme concentration, pH >9.0, high temperature) that limit its applicability.
• Current findings suggest limitations for using proteolytic methods on delicate surgical instruments or in environmental decontamination due to harsh requirement.