With the emergence of multi-drug resistant (MDR) “super bugs” the need for new bactericidal approaches is growing in urgency. Compounding the threat of MDR, is the rise of antibiotic resistance in weaponizable pathogens such as Bacillus anthracis.

Anthrax, caused by Bacillus anthracis, remains a cause for global concern, even if memories of the 2001 acts of bioterrorism where mail containing anthrax-causing spores were sent to media outlets and members of Congress, may be fading. The “Amerithrax” attacks killed five and sickened at least 22 people.

Back in the 1930s pioneers in molecular biology and genetics had used enzymes that degrade bacterial capsules to combat infections. While capsules enveloping bacteria are usually composed of polysaccharides, the capsule of virulent strains of Bacillus anthracis is made of polymers of D-glutamic acid (PDGA) linked covalently by gamma-glutamyl bonds.

A new study published in the journal ACS Infectious Diseases “Engineering an Fc-fusion of a Capsule Degrading Enzyme for Treatment of Anthrax” on September 14. 2022,  reports the development of an  engineered hydrolase enzyme called CapD that is modified and enhanced to degrade the outer protective capsule of Bacillus anthracis in culture dishes and in mouse models, without the aid of antibiotics, vaccines or antibodies.

Patricia M. Legler, PhD, a research biologist focusing on structural enzymology and protein engineering in the Center for Bio/Molecular Science and Engineering at the US Naval Research Laboratory is the senior author of the study [US Navy photo by Jonathan Steffen]

“The investigation was inspired by growing concerns over the emergence of drug-resistant pathogens,” said the senior author of the study, Patricia Legler, PhD, currently a scientist at the US Naval Research Laboratory, who previously worked at the Walter Reed Army Institute of Research. “These threats impact global health, food security, and can lead to higher mortality rates and increased medical costs. Also, we wanted to reexamine research into the use of enzymes to treat infections that was pioneered in the 1930s but was largely abandoned after the advent of antibiotics.”

This engineered CapD enzyme with bits of antibody stuck to it can break apart the capsule of B. anthracis bacteria, allowing the immune system to fight back [Adapted from ACS Infectious Diseases, Matharoo et al, 2022]

The study reports the challenges and solutions in engineering a soluble, recombinant, polyethylene glycol-attached fusion of CapD with the crystallizable fragment (Fc) of a mouse antibody in Eschericia coli that is stable at both acidic and neutral pH and effective in combating infection in vitro and in vivo.

Spores of Bacillus anthracis can cause anthrax infection when they enter the body either by ingestion, inhalation or through open wounds. Anthrax infection can lead to difficulty breathing, skin ulcers and even death. Antibiotics against anthrax infection are available, such as ciprofloxacin and doxycycline, but resistance to them occur over time. The Ames strain of Bacillus anthracis is particularly virulent because of its protective capsule that acts like an invisibility cloak, enabling the bacillus to evade the host’s immune system.

CapD in Bacillus anthracis normally anchors the bacteria’s PDGA capsule. Earlier studies had suggested that the enzyme could be engineered to degrade the capsule instead. Once the invisibility cloak of the capsule is degraded, the bacteria can be seen by the host’s immune system, which goes on to mount an attack against the pathogen.

Earlier studies had also shown, treatment of mice with an engineered CapD can ameliorate Ames-strain anthrax infections. Previous studies by Legler and colleagues had demonstrated, adding polyethylene glycol (PEG) to the engineered version of CapD induced the enzyme to stick around longer, consequently increasing survival of mouse models.

In the current study, Legler’s team optimized CapD further to increase the enzyme’s active lifespan in circulation. To deliver a bigger punch, the researchers added PEG and fused CapD with the crystallizable part of a mouse antibody (Fc). This linked two CapD molecules into a dimer that effectively doubled its ability to bind to the bacterial capsule. To accomplish this enhancement of CapD, the researchers created several versions of the enzyme and subjected each to many rounds of optimization, deleting and inserting different segments until they achieved a 3D configuration that performed effectively at a range of pH values.

“This is the first Fc-fusion of CapD to be described. Therapeutic enzymes for bacterial pathogens have only recently entered into clinical trials. The first to enter a clinical trial was a lysin for the treatment of MRSA (NCT04160468) by Contrafect Inc. Although testing of enzyme extracts to treat bacterial infections actually dates back to the 1930s, enzymes are only now entering into clinical trials for multidrug-resistant bacterial therapeutics, and others will likely be developed,” said Legler.

When tested in a mouse model, the engineered construct lasted longer in circulation than the previous version without the fused antibody, although its catalytic activity was slightly reduced.

“We have shown that the pegylated capsule depolymerase, CapD, can protect against five LD50 challenges with lethal Ames spores in a mouse model study. We have also developed a novel Fc-fusion of CapD,” said Legler. LD50 is a parameter commonly used in toxicology to denote 50 percent of the lethal dose or the amount of the toxin needed to kill half of all test organisms.

The findings are an important step to combating antibiotic-resistant strains of Bacillus anthracis, although further studies are needed to produce the ideal construct.

Legler noted, “We plan to test and optimize the pegylated CapD enzyme in mouse studies and in a rabbit study in the future. Right now, we are testing a variety of CapD variants to see which is the most stable and effective in vivo.”

The Defense Threat Reduction Agency funded this investigation.