University of Illinois chemist Wilfred van der Donk (right) has identified a chemical compound that could be the next frontier in mastitis treatment. The synthetic compound, geobacillin, is similar to a natural mastitis-fighting compound, nisin, which is made in a cow’s udder. A commercial form of nisin for mastitis treatment is currently under FDA review and a decision is expected soon. (Read an update about this drug’s approval status below.) Research initially shows the new compound to be even more potent than nisin against at least one bacterial strain that causes mastitis. Progressive Dairyman Editor Walt Cooley talked with Van der Donk about his discovery.
Q. Tell me a little bit about your background. Did it previously touch the dairy industry?
A. VAN DER DONK: No, not really. However, we have been investigating how nisin is made by its producing-bacteria called Lactococcus lactis – that’s a bacterium that lives in the udder of a cow. It was actually in milk, where nisin was discovered back in 1927.
Our interest in nisin stems back 10 years, and we have worked a lot on the enzymes that make nisin. It turns out that for a chemist/microbiologist/biochemist, the way nature makes nisin is really, really fascinating.
My background is in chemistry, but my lab is also full of microbiology students and biochemistry students. We really work at the interface between chemistry, biochemistry and microbiology.
Q. When did you first get interested in nisin?
A. VAN DER DONK: When I was a post-doc and I was looking for projects to work on in my own lab, I came across nisin back in 1997. I was intrigued by its structure but even more intrigued by the manner in which it was made in nature. When I started here at the University of Illinois in the fall of 1997, we started a program trying to see if we could make nisin in a test tube by expressing and purifying the enzymes that normally make nisin in Lactococcus lactis.
Nisin is made by one enzyme that breaks 16 chemical bonds, and then a second enzyme that creates 10 new chemical bonds. As a chemist, I am in awe as to how an enzyme can do all of that. Nisin was synthetically made by chemical means in the ’70s and it took chemists 67 chemical steps in order to make nisin.
In nature, it takes just two enzymes and boom-boom you have nisin. We always thought that we might be able to make analogues (a compound with a molecular structure closely similar to that of another) of nisin that perhaps could be better than nisin itself through better understanding the enzymes that make nisin. That has been one project of my lab.
Q. What are nisin’s uses?
A. VAN DER DONK: Nisin has been used for more than 40 years now. Again, it’s made by the lactic acid bacterium Lactococcus lactis at an acidic pH of 4 to 5. So for many years, Lactococcus lactis or nisin has been added to foods that have acidic pHs to prevent the growth of food-borne pathogens, especially listeria and clostridia.
Remarkably, over time, very little resistance has been developed among the pathogens nisin is intended to kill, which is unusual for a compound with that widespread use.
Nisin went into pre-clinical trials in the ’80s to see if the molecule could also be useful to treat infections in humans. It became clear that it was very, very potent; it killed pathogens in our blood really fast. However, in our blood, where the acidity is neutral, nisin appears to break down quickly. Ever since, we’ve worked towards making a compound like nisin that is potent but more stable.
Q. So what did you do in your research to pursue this goal?
A. VAN DER DONK: We were scanning the bacteria genome databases for genes that we thought might make nisin analogues. Our thinking was that if we could find an organism that produces nisin or a nisin analogue, and it grows in non-acidic, high-temperature conditions, then most likely the compound will be stable in non-acidic conditions and lower temperature conditions too.
So once we found the genes we were looking for, we put them in E. coli bacteria. We were asking E. coli to help us to make the molecules similar to nisin, and that worked. E. coli made the molecule for us. We studied its structure and determined indeed it was a nisin analogue and that it had better stability than nisin.
Q. What do you call this compound?
A. VAN DER DONK: Geobacillin
Q. Why use E. coli to grow an analogue? Isn't E.coli bad?
A. VAN DER DONK: E. coli is the bacterium that is best understood by humans, and it’s so well understood that E. coli strains have been created that are non-pathogenic, non-dangerous. Those are the strains that are used all over the globe in many, many different laboratories, both in academia and also in industry, to make useful enzymes.
We used an engineered E. coli strain that everybody has been using for decades, one that the scientific research community understands the biology very, very well.
We wanted E. coli to express as the genes from a thermophilic organism, one that grows at 70 degrees Celsius or 158 degrees Fahrenheit. We know how to manipulate E. coli so that we can make a lot of the enzymes.
So just by growing a few liters of E. coli with the right genes inserted, we made plenty of the desired nisin analogues, in this case geobacillin. We did this to determine geobacillin’s structure, to look at its mode of action and to look at its activity against various pathogens.
Q. What are the similarities and differences between the geobacillin you made and the naturally occurring nisin?
A. VAN DER DONK: Nisin is a peptide that is made out of 34 amino acids that are strung together. A peptide is a bit like spaghetti; it has no restriction to be in a certain form or another; it can be in all kinds of different shapes. A peptide is very floppy. If you want to recognize a certain target for a peptide, say, something that will kill a bacterium that causes bovine mastitis, you want your peptide not to be floppy.
You want it to be made only in that form where it can bind to the target. So what nature has come up with are all kinds of rings in peptides. Think about it as putting ties in your spaghetti so that now it’s much less floppy.
The peptide nisin has five rings in it. The first two rings bind to something that is absolutely essential for all bacteria to make a cell wall. Why is that important? Bacteria need cell walls. If they don’t have cell walls, they burst – literally. So if you prevent a bacterium from making a cell wall, it will die.
The last three rings in nisin poke holes in the bacterium’s membrane, which leads to the loss of nutrients. The double-edged mode of action on a bacterium of preventing it from protecting itself and, at the same time, poking holes in it, is what makes nisin so great.
That’s what makes nisin kill bacteria in such low concentrations. The difference between geobacillin and nisin is that the geobacillin has two more rings than nisin – it has seven. Those two additional rings make the compound even more stable, less floppy.
Q. Your research showed geobacillin was three times more effective against a main form of mastitis-causing bacteria (Streptococcus dysgalactia)?
A. VAN DER DONK: Yes, that’s correct, although when we do that test, it is growing this bacterium in isolation and exposing it to geobacillin, of course. Note that we say the main contagious agent; there are many different bacteria that cause mastitis. We didn’t test all of them; we just picked one.
It gets much more complex when an organism is growing inside the udder of a cow. You cannot extrapolate directly that these results would necessarily translate to this compound also being threefold better in the treatment of bovine mastitis. That is certainly a possibility, but there is no guarantee.
Most people would say that the more exciting part of geobacillin is its stability – the stability at higher pH and the stability at higher temperature that might allow this compound to be really interesting. Only time will tell.
Q. So you synthetically created a bacteria-fighting compound similar to nisin but that was more stable – why is that a breakthrough?
A. VAN DER DONK: We became really interested in potential applications of geobacillin. It was then that we found out about the pending use of nisin in the dairy industry as a treatment of bovine mastitis. As I understand it, ImmuCell has the idea that if you treat bovine mastitis infection with nisin, then all you are doing is adding nisin to the cow’s biological system, and nisin is already in the udder of a cow anyway. It’s in milk already.
That pharmaceutical drug use of nisin is pending final FDA approval.* So if you treat bovine mastitis with nisin, you would not have to discard your milk, which is currently the case if you treat with other antibiotics. ImmuCell’s drug application filing requests that nisin use be approved with a zero-discard time, and you might even be able to sell a cow for meat during the time that you treat with nisin.
So we became interested in what organisms cause bovine mastitis and whether our synthetic compound could kill those organisms, too.
Q. So if nisin becomes FDA approved for use in milking cows, could your analogue, geobacillin, eventually be used as a treatment too?
A. VAN DER DONK: Yes, I think it has that potential. However, there are many, many things that we know about nisin that would have to be confirmed about geobacillin. There’s a long road ahead of us, but the fact that it looks so similar to nisin and has all of the hallmarks of nisin shows it has that potential.
Certainly, I think FDA approval of nisin would be a great step forward. If I were a dairy farmer, I would see this as a great improvement in losses that would no longer be incurred when a cow has mastitis.
Q. Any closing thoughts about this breakthrough?
A. VAN DER DONK: We’re excited about it, and we certainly will look into whether the potential advantages of this compound, which currently has all the hallmarks of being an improved version of nisin, will indeed hold true. We are going to look at all of its different applications. PD
*NOTE: ImmuCell’s Mast Out, an intramammary infusion product that contains the antibacterial peptide nisin, is currently under FDA review. Commercial introduction of the product in the U.S. is subject to approval of the company’s New Animal Drug Application (NADA). The company has been the sponsor of the investigational NADA from 2001-2004 and from 2007 to present. Several key regulatory approval decisions about the potential product are expected this year. FDA’s Center for Veterinary Medicine has granted the company a “zero-milk discard and meat withhold” label claim that would permit use of its product in lactating cows and not require milk be withheld or treated animals to be sold for meat, pending full NADA approval. The company asserts it would be the first FDA-approved intramammary mastitis treatment product that has such a claim.
Walt Cooley
Editor
Progressive Dairyman
