Quelling Pathogens with Engineered Proteins

AvidBiotics Leverages DGR Platform to Harness Killing Mechanism of R-Type Pyocins

After David Martin, M.D., read an article in Nature in 2004 about the discovery of diversity-generating retroelements (DGR), he scheduled a meeting with the researcher, Jeffery Miller, Ph.D., at the University of California, Los Angeles. Their discussions led to the founding of AvidBiotics in 2005 to advance DGR technology. “DGR technology is a broad platform for protein engineering,” says Dr. Martin, CEO of AvidBiotics.

DGRs are a family of genetic elements in prokaryotic organisms and bacteriophages that diversify DNA sequences and the proteins they encode. The prototype DGR was identified in the bacteriophage BPP-1 that infects Bordetella species, and about 100 more have been found in other microbes.

The action of DGRs resembles that of antibodies and T-cell receptors in the immune system, which recognize trillions of foreign molecules and generate precise antibodies against them. Similarly, DGRs can generate trillions of variations of a protein molecule. AvidBiotics’ goal is to create novel molecules to kill specific bacterial or viral pathogens without harming beneficial microbes.

Researchers at AvidBiotics are applying the DGR platform to R-type pyocins, which are proteins made by one bacterium to defend itself against other bacteria. The company’s Avidocin™ proteins are based on engineered R-type pyocins that target and kill clinically important pathogens, such as Escherichia coli and Clostridium difficile.

Avidocin proteins offer several advantages over traditional antimicrobial therapies, according to Dr. Martin. R-type pyocins are pure proteins and highly focused antibacterials. One pyocin kills one bacterium, and they are extremely potent. By targeting just one bacterial pathogen, pyocins spare healthy microbial communities from collateral damage.

Unlike antibiotics, pyocins are insensitive to methods bacteria use to resist antibiotics. And unlike bacteriophages, pyocins do not contain nucleic acids and cannot replicate inside targeted cells. Additionally, Dr. Martin says, Avidocin proteins can be manufactured by using common fermentation processes.

Avidocin proteins are designed and engineered to kill specific bacteria by attacking them on exposed surface molecules that convey pathogenicity. Researchers fuse a tail fiber gene of a bacteriophage (known to attack a particular bacterium) onto the pyocin tail fiber gene, which then redirects the engineered protein to a targeted bacterium.

After binding tightly via their tail fibers to the bacterial surface, pyocins plunge their needle-like core into the target bacterium. Penetration depolarizes the bacterial membrane and rapidly kills the pathogen. This method has generated Avidocin proteins that kill strains of E. coli, Salmonella, Shigella, and Yersinia pestis.

AvidBiotics is also applying its technology to food safety. “We engineered a pyocin that kills only E. coli O157/H7,” Dr. Martin says. Collaborators at the U.S. Department of Agriculture have tested this engineered pyocin on their extensive collection of pathogens. The plan is to spray the Avidocin protein on animal carcasses or trim (the precursor of hamburger) at meat-processing plants. Laboratory experiments predict that treating an entire beef carcass with just 10 milligrams of the pyocin should kill significant numbers of E. coli O157/H7.

In January, AvidBiotics and Ecolab inked an agreement to develop the technology to reduce E. coli O157/H7 in red meat products. E. coli O157/H7 infects more than 70,000 people yearly in the U.S., according to the Centers for Disease Control and Prevention.

“Combining AvidBiotics’ technology and expertise in molecular biology with Ecolab’s technical competencies and market position will allow us to accelerate the path to commercialization,” Dr. Martin says. Ecolab is involved in cleaning, sanitation, food safety, and infection protection. “Its commitment to collaborate with us validates our approach to combat deadly pathogens.”

Antimicrobials for Stubborn Infections

AvidBiotics is also pursuing an Avidocin treatment for C. difficile, a common hospital-acquired infection. About 3% of patients carry C. difficile in their intestinal tract when they enter a hospital, but after a stay of four weeks or longer, half of all patients become carriers of C. difficile.

R2 pyocins illustration
R2 pyocins are pure proteins and highly focused antibacterials (left). Avidocin R2-V10 (right) is based on engineeered R-type pyocins that target and kill pathogens such as E. coli and C. difficile.

When carriers are treated with antibiotics, this antibiotic-resistant pathogen flourishes, causing severe diarrhea and colitis. “We have developed an R-type pyocin that kills C. difficile and only C. difficile,” says Dr. Martin.

He foresees treating patients with the protein agent during their entire hospital stay to eliminate the pathogen from the intestines. “You can’t do this with any other antimicrobial drugs because they kill beneficial bacteria as well.”

Recurrent urinary tract infections (UTIs) are another target for Avidocin proteins. Strains of E. coli often cause recurrent urinary tract infections, and “we have Avidocin proteins that kill uropathogenic E. coli in the colon,” says Dr. Martin. While not pathogenic in the colon, E. coli becomes pathogenic when it reaches the bladder. “The idea is to administer the Avidocin protein to eliminate E. coli in the colon before it reaches the bladder.” The Avidocin proteins for C. difficile and UTIs will likely be given orally.

Future Plans

Drs. Martin and Miller, and James Knighton co-founded AvidBiotics to pursue therapies for microbial diseases. Reliable in vitro and animal models make it easy to validate targets and evaluate therapeutic candidates early on and relatively inexpensively.

Rather than risking investor funds, “we bootstrapped the operation largely with monies from friends, family, and founders, and three SBIR grants,” Dr. Martin says.

If their DGR hunch pans out, they will seek venture capital funds needed to support further development of human therapeutics.