Engineering R&D: Engineered bacteria killers
As understanding of how nature adapts to the evolution of harmful microorganisms deepens, molecular biology is delivering new food safety tools, including bactericidal proteins.
Biotechnology and genetic manipulation seems far removed from carcass washing in a packinghouse, but a recent collaboration between Ecolab Inc. and a South San Francisco, CA, biotherapeutics firm could yield a new weapon against food-borne pathogens, beginning with E. coli O157:H7. In January, Ecolab and Avid Biotics Corp. announced the development of Avidocin, a bactericidal protein engineered to attack pathogens of concern in red meat.
Researchers at the University of California at Los Angeles’ microbiology, immunology and molecular genetics lab developed Avid Biotics’ core technology, which uses a nucleic acid molecule as a template for diversification in proteins, allowing them to adapt as the life forms they are tailored to attack also adapt and mutate. Avid Biotics originally focused on development of drugs that could replace antibiotics rendered useless by “superbugs” that evolve to resist these antibiotics. These nucleic acid molecules are very similar to bacteriophage, a type of virus that targets specific bacteria. (See “Bacteria busters,” Food Engineering, September 2007.) The company’s name is a play on avidity, a term describing the combined strength of multiple bond interactions, in this case six pods attached to a protein scaffold.
Since its founding in 2005, Avid Biotics’ development has been guided by two men: CEO David Martin, a professor of medicine and biochemistry and researcher at several pharmaceutical companies; and James L. Knighton, president. As an MBA candidate at University of Pennsylvania’s Wharton School, Knighton was drawn to developments in biotechnology and became the first student to receive a master’s degree in genetics from the business school. He has been involved in R&D, marketing and finance in several organizations, including DuPont Merck, Chiron, Sugen and Caliper Life Sciences, which he helped guide through its initial public offering as president. Food Engineering recently spoke with Knighton about Avid Biotics technology and its potential use in food production.
FE: How does your bactericidal agent differ from bacteria-killing viruses known as bacteriophage?
Knighton: They are close cousins. Both attack specific bacteria; both have tremendous diversity to change as the bacteria they target mutates to get away. How the phage do that is one of the most elegant mechanisms we have ever seen and involves trillions of different combinations until it finds a solution. The difference is that we engineer R-type pyocins, which are high-molecular weight phage tail-like protein complexes that cannot reproduce. Phage inject DNA into a bacterium, using the host as an incubator until the bacterium bursts, releasing more phage until the target population is wiped out. With R-type pyocins, there is single-hit kinetics: one pyocin produces one dead bacterium. That means you can dose it, and the FDA loves that for therapeutic drugs.
FE: How do you engineer the pyocins?
Knighton: We begin by finding a bacteriophage that kills the target organism, then clip off the tail tip of its pods and attach it to the pyocin tail fiber gene. When some of the pyocin’s six pods attach to E. coli, Salmonella or Yersinia pestis, they do not seal the hole they create, unlike a phage. Instead, vital chemicals leak out through the pyocin’s hollow core, resulting in the target organism’s death.
FE: The core technology is referred to as diversity generating retroelements (DGR). How does that relate to R-type pyocins?
Knighton: DGR is confusing to our investors and, even though it was our founding technology, it is not used to modify R-type pyocins used in food systems. Instead, this is more dependent on a ligand-binding protein scaffold that was discovered by researchers at the University of San Diego. It is used to generate functional binding diversity.
A scaffold is simply a structure for the protein. Think of it as a mannequin that supports the protein that you then modify to attack the desired bacterium. This particular scaffold accommodates 10 trillion sequences, making it almost as adaptable as the human immune system.
FE: How did the technology migrate from therapeutics and diagnostics to food safety applications?
Knighton: Rob Mandrell, a biochemist with USDA’s Agricultural Research Service, contacted us. He had E. coli O157:H7 isolates from 58 food poisoning outbreaks, ranging from Jack in the Box in 1993 to spinach harvested in Salinas, CA, in 2006. He also had some E. coli from non-O157:H7 outbreaks. We fused a tail spike protein from an O157-specific phage to the pyocin tail fiber, and the tail-spike protein recognized and killed the pathogen isolates but no other serotypes.
R-type pyocin is highly specific, and that can be valuable. Clostridium difficile is a serious hospital infection affecting the gastrointestinal tract. Antibiotics wipe out competing, good bacteria in the GI tract, resulting in diarrhea and other intestinal disease. Killing C-difficile while leaving the good bacteria alone can save lives.
By the same token, many foods have good bacteria, which is why Ecolab approached us. We successively demonstrated this engineered pyocin’s effectiveness as an antimicrobial agent on beef surfaces in the lab. We hope to test it in a plant this year.
FE: How receptive is industry to this intervention?
Knighton: We have visited producers such as Tyson and Dole. They are focused on the added cost. We also have talked with firms such as Costco and McDonald’s. Their view of acceptable cost is a different cat altogether, and the reaction was very positive. We also have talked with some food advocacy groups, who are dead set against the use of phage or irradiation.
FE: Since 2006, FDA has given GRAS status to several phage cocktails that attack particular food pathogens. Why is development of an alternative antimicrobial needed?
Knighton: This isn’t about science, it’s about commerce, and the two don’t always go together. In industry jargon, the phrase “Mom won’t buy it” is applied to phage. Market research says phage won’t pass muster with consumers. “Mom doesn’t like blowing DNA on bologna.” Companies may not have to declare the use of phage as an antimicrobial, but all it takes is one food advocacy group to flag its use, and you’re dead. Phage will not affect humans, so you may win the battle, but you’ve already lost the war.
FE: Biodefense is another application you’re exploring. How does the technology serve those objectives?
Knighton: Yersinia pestis is the bacterium linked to the spread of bubonic plague. If Y-pestis was weaponized in an aerosol form, it would be harder to treat with antibiotics than the plague. We received a second small business innovation grant last year to develop R-type pyocins to kill Y-pestis.
A group from New Zealand visited us. They thought there was an application in fighting greenhouse gases (GHG) generated by livestock, which account for approximately 18 percent of all GHG. Methanogen bacteria create livestock’s methane. If we could develop a bactericidal protein that only killed 20 percent of methanogens without disturbing other bacteria in the gut, the New Zealanders think we’d have a winner.
FE: How large is the Avid Biotics staff?
Knighton: Seven people as I speak, and we’re in the process of hiring our eighth person. Our head of research is a phage biologist, incidentally.
The company was founded with a core group of three: Jeffery Miller, a professor of microbiology, immunology and molecular genetics at UCLA who has gone back to the lab; Dave Martin, a world class scientist and a former R&D executive at Genentech and DuPont Merck; and me. I had been retired for a couple of months when Dave, who saw great potential in the technology, called me and said, “Let’s start a company.”
The human immune system relies on antibodies with the ability to attack quadrillions of antigens through the adaptability of the antigen-binding site. The same adaptability is being grafted onto engineered bactericidal proteins that attack food-borne pathogens.
Biotechnology and genetic manipulation seems far removed from carcass washing in a packinghouse, but a recent collaboration between Ecolab Inc. and a South San Francisco, CA, biotherapeutics firm could yield a new weapon against food-borne pathogens, beginning with E. coli O157:H7. In January, Ecolab and Avid Biotics Corp. announced the development of Avidocin, a bactericidal protein engineered to attack pathogens of concern in red meat.
Researchers at the University of California at Los Angeles’ microbiology, immunology and molecular genetics lab developed Avid Biotics’ core technology, which uses a nucleic acid molecule as a template for diversification in proteins, allowing them to adapt as the life forms they are tailored to attack also adapt and mutate. Avid Biotics originally focused on development of drugs that could replace antibiotics rendered useless by “superbugs” that evolve to resist these antibiotics. These nucleic acid molecules are very similar to bacteriophage, a type of virus that targets specific bacteria. (See “Bacteria busters,” Food Engineering, September 2007.) The company’s name is a play on avidity, a term describing the combined strength of multiple bond interactions, in this case six pods attached to a protein scaffold.
Since its founding in 2005, Avid Biotics’ development has been guided by two men: CEO David Martin, a professor of medicine and biochemistry and researcher at several pharmaceutical companies; and James L. Knighton, president. As an MBA candidate at University of Pennsylvania’s Wharton School, Knighton was drawn to developments in biotechnology and became the first student to receive a master’s degree in genetics from the business school. He has been involved in R&D, marketing and finance in several organizations, including DuPont Merck, Chiron, Sugen and Caliper Life Sciences, which he helped guide through its initial public offering as president. Food Engineering recently spoke with Knighton about Avid Biotics technology and its potential use in food production.
James L. Knighton, president, Avid Biotics Corp., South San Francisco, CA. Source: Avid Biotics Corp.
Knighton: They are close cousins. Both attack specific bacteria; both have tremendous diversity to change as the bacteria they target mutates to get away. How the phage do that is one of the most elegant mechanisms we have ever seen and involves trillions of different combinations until it finds a solution. The difference is that we engineer R-type pyocins, which are high-molecular weight phage tail-like protein complexes that cannot reproduce. Phage inject DNA into a bacterium, using the host as an incubator until the bacterium bursts, releasing more phage until the target population is wiped out. With R-type pyocins, there is single-hit kinetics: one pyocin produces one dead bacterium. That means you can dose it, and the FDA loves that for therapeutic drugs.
FE: How do you engineer the pyocins?
Knighton: We begin by finding a bacteriophage that kills the target organism, then clip off the tail tip of its pods and attach it to the pyocin tail fiber gene. When some of the pyocin’s six pods attach to E. coli, Salmonella or Yersinia pestis, they do not seal the hole they create, unlike a phage. Instead, vital chemicals leak out through the pyocin’s hollow core, resulting in the target organism’s death.
FE: The core technology is referred to as diversity generating retroelements (DGR). How does that relate to R-type pyocins?
Knighton: DGR is confusing to our investors and, even though it was our founding technology, it is not used to modify R-type pyocins used in food systems. Instead, this is more dependent on a ligand-binding protein scaffold that was discovered by researchers at the University of San Diego. It is used to generate functional binding diversity.
A scaffold is simply a structure for the protein. Think of it as a mannequin that supports the protein that you then modify to attack the desired bacterium. This particular scaffold accommodates 10 trillion sequences, making it almost as adaptable as the human immune system.
FE: How did the technology migrate from therapeutics and diagnostics to food safety applications?
Knighton: Rob Mandrell, a biochemist with USDA’s Agricultural Research Service, contacted us. He had E. coli O157:H7 isolates from 58 food poisoning outbreaks, ranging from Jack in the Box in 1993 to spinach harvested in Salinas, CA, in 2006. He also had some E. coli from non-O157:H7 outbreaks. We fused a tail spike protein from an O157-specific phage to the pyocin tail fiber, and the tail-spike protein recognized and killed the pathogen isolates but no other serotypes.
R-type pyocin is highly specific, and that can be valuable. Clostridium difficile is a serious hospital infection affecting the gastrointestinal tract. Antibiotics wipe out competing, good bacteria in the GI tract, resulting in diarrhea and other intestinal disease. Killing C-difficile while leaving the good bacteria alone can save lives.
By the same token, many foods have good bacteria, which is why Ecolab approached us. We successively demonstrated this engineered pyocin’s effectiveness as an antimicrobial agent on beef surfaces in the lab. We hope to test it in a plant this year.
FE: How receptive is industry to this intervention?
Knighton: We have visited producers such as Tyson and Dole. They are focused on the added cost. We also have talked with firms such as Costco and McDonald’s. Their view of acceptable cost is a different cat altogether, and the reaction was very positive. We also have talked with some food advocacy groups, who are dead set against the use of phage or irradiation.
FE: Since 2006, FDA has given GRAS status to several phage cocktails that attack particular food pathogens. Why is development of an alternative antimicrobial needed?
Knighton: This isn’t about science, it’s about commerce, and the two don’t always go together. In industry jargon, the phrase “Mom won’t buy it” is applied to phage. Market research says phage won’t pass muster with consumers. “Mom doesn’t like blowing DNA on bologna.” Companies may not have to declare the use of phage as an antimicrobial, but all it takes is one food advocacy group to flag its use, and you’re dead. Phage will not affect humans, so you may win the battle, but you’ve already lost the war.
FE: Biodefense is another application you’re exploring. How does the technology serve those objectives?
Knighton: Yersinia pestis is the bacterium linked to the spread of bubonic plague. If Y-pestis was weaponized in an aerosol form, it would be harder to treat with antibiotics than the plague. We received a second small business innovation grant last year to develop R-type pyocins to kill Y-pestis.
A group from New Zealand visited us. They thought there was an application in fighting greenhouse gases (GHG) generated by livestock, which account for approximately 18 percent of all GHG. Methanogen bacteria create livestock’s methane. If we could develop a bactericidal protein that only killed 20 percent of methanogens without disturbing other bacteria in the gut, the New Zealanders think we’d have a winner.
FE: How large is the Avid Biotics staff?
Knighton: Seven people as I speak, and we’re in the process of hiring our eighth person. Our head of research is a phage biologist, incidentally.
The company was founded with a core group of three: Jeffery Miller, a professor of microbiology, immunology and molecular genetics at UCLA who has gone back to the lab; Dave Martin, a world class scientist and a former R&D executive at Genentech and DuPont Merck; and me. I had been retired for a couple of months when Dave, who saw great potential in the technology, called me and said, “Let’s start a company.”
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