Source: United States Navy
The team of researchers detailed their research findings funded by the Defense Threat Reduction Agency (DTRA) in an article published Dec. 8 in Science Translational Medicine.
Anthrax is a severe infectious disease caused by gram-positive, rod-shaped bacteria known as Bacillus anthracis. According to the authors, it is also considered one of the most dangerous bioweapon agents.
While there are vaccine and antibiotic therapies available for anthrax, the rise of multidrug-resistant strains of the disease is a growing concern.
Looking to the past to inspire innovative ideas, the researchers focused on a method first pioneered in the 1930s to use enzymes to treat infections. In particular, the team developed an engineered enzyme to target the anti-phagocytic capsule of Bacillus anthracis. While vaccine strains of B. anthracis (e.g., Sterne Strain) are unencapsulated and can be removed by the human immune system, strains like the lethal Ames strain have a capsule.
Leading the project for NRL is Patricia Legler, Ph.D., senior scientist in chemical and biodefense at the Center for Biomolecular Science and Engineering.
“The capsule is like a “cloak of invisibility” so your immune system doesn’t see the encapsulated bacteria,” Legler said. “In the blood it can grow and cause lethal septicemia.”
Co-author Arthur M. Friedlander, M.D., senior scientist at USAMRIID, postulated that the enzyme CapD had the potential to be used as a therapeutic.
Legler served as the team’s enzymologist to develop a highly active form of the CapD enzyme. To accomplish this, she used a method called PEGylation. In PEGylation, the biological molecules are modified by covalent conjugation with polyethylene glycol (PEG), a non-toxic, non-immunogenic polymer.
“The PEG molecule wraps around the enzyme much like an octopus binding its prey,” Legler said.
Using kinetic and biophysical methods, Legler was able to create a stable and active PEGylated enzyme in high yield that could unencapsulate the anthrax bacteria and allow the innate immune system to clear the bacteria. Adding the PEG to the wrong location on the enzyme can block the active site and end all enzyme activity.
PEGylation accomplished both raising the temperature tolerance and the sustainability of the enzyme in vivo.
“When proteins are heated, they tend to unfold and aggregate irreversibly,” Legler said. “With the PEG, I was able to elevate the melting temperature of the enzyme by about 7 degrees. This enabled the enzyme to withstand the 37 degrees Celsius body temperature for long periods.”
Legler’s PEGylated enzyme allowed the team to evaluate the success of the enzyme treatment in a mouse model using the lethal Ames strain of the bacteria.
Based on the results of the rodent testing, protection levels were achieved solely with enzyme treatment without the addition of vaccines or antibiotics.
“Engineered threats or naturally developed threats are of concern,” Legler said. “Most people aren’t vaccinated for B. anthracis, and inhalation anthrax is highly lethal. By developing enzyme therapies, we can potentially treat these types of threats and eliminate them.”
Besides the demonstrated efficacy of the treatment, it is also noteworthy that the research is one of the first to successfully produce and test enzyme therapies in vivo for bacterial infections.
Looking forward, the team will investigate applying their therapeutic enzyme approach to other bacterial pathogens.
“Now, with the wealth of molecular biology tools, we can engineer a variety of different enzyme therapeutics,” Legler said. “These ‘biologics’ are novel and have not been tested in vivo. Adding these novel therapies to our arsenal is valuable because they differ from antibiotics.”
About the U.S. Naval Research Laboratory
NRL is a scientific and engineering command dedicated to research that drives innovative advances for the U.S. Navy and Marine Corps from the seafloor to space and in the information domain. NRL is located in Washington, D.C. with major field sites in Stennis Space Center, Mississippi; Key West, Florida; Monterey, California, and employs approximately 3,000 civilian scientists, engineers and support personnel.
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