A few times a year Stan Brouns (1978) goes to the wastewater treatment plant to collect some samples. He understands that for many people it is not a pleasant idea to bring home buckets of sewage, but for him it is a goldmine. “Each of us has half a kilo of bacteria in our intestines. That’s billions. Some of it ends up in the wastewater. These samples not only contain countless variants of human bacteria, but also of their natural enemies, the bacteriophages,” says the professor of microbiology at TU Delft. “Last year we discovered a whole new family of phages, which we named after that famous Delft resident, Antoni van Leeuwenhoek.”
Brouns sees a medical application in these natural enemies: fighting bacterial infections. A doctor usually resorts to an antibiotic for this, but some bacteria have become resistant to this panacea. In that case, a bacteriophage can take over the battle. But that is not yet allowed in the Netherlands. The efficacy and safety of phage therapy has not yet been clinically proven. Until then, Brouns is already building a database of bacteriophages of which he knows how effective they can be. His collection now contains 150 copies.
This week it was announced that Brouns is one of the eight winners of the Ammodo Science Award. The prize is a recognition for his scientific research. Brouns will use the corresponding amount of money, 350,000 euros, to expand his phage collection.
Every day, bacteriophages kill a third of all bacteria in the ocean
Bacteriophages are a group of viruses that target bacteria. Every day, bacteriophages kill a third of all bacteria in the oceans. But the phages are very picky. There are many types of bacteria, and each type has many variants. But each bacteriophage can only infect one type of bacteria, or at most a few variants.
They look like a lunar lander, says Brouns. A capsule on long legs with a tail between them. Brouns: “Once the phage has found a suitable bacterium, landed on it and everything fits, it inserts its tail and injects its DNA into the host cell under high pressure. It pops like a champagne bottle. The DNA of the phage takes over the machinery of the bacterial cell and makes as many copies of itself as possible. If that succeeds, the bacteria will die.”
It has been known for more than a century that bacteriophages can be used against bacterial infections. In many countries the therapy fell into oblivion due to the emergence of antibiotics, but in Georgia it is still standing practice and in Belgium and Poland, for example, phages are on the rise again. The major drawback is that they are so specific. You cannot use them for an acute infection, says Brouns. “You will first have to find out on a petri dish which bacteriophage is effective. You only do that in the case of a chronic infection that no antibiotics seem to work against.”
Efficacy is difficult to prove
A second disadvantage is that their effectiveness is difficult to prove. A large clinical trial, in which half of the patients with an infection receive a bacteriophage and the other a placebo, is doomed to fail. These patients all have variants of a bacterium that cannot be combated with one and the same bacteriophage. It is still impossible to put together a group with all exactly the same bacteria, says Brouns.
In the meantime, there are many studies that advocate bacteriophages. Descriptions of individual patients who have been cured with the help of phages convinced Brouns himself. But he leaves that matter to the doctors. “The scientific explanation for their efficacy and safety is strong, but we are not concerned with the clinical evidence. We just build that collection. The aim is to find a series of phages for the ten or so bacterial species that pose a problem in hospitals due to their resistance.”
In his fundamental research, he focuses more on the bacterium: how does it defend itself against the bacteriophage? The two microorganisms have been engaged in an arms race for billions of years. The phage attacks, the bacteria develops a defense, which the phage then tries to avoid. It’s a life and death struggle. The bacteria only have a few minutes to recognize and fight the virus DNA.
Defense mechanisms
In this age-old evolution, the bacterium has acquired all kinds of defense mechanisms to keep the phages at bay. Those lines also hinder phage therapy. Brouns recently received a large European subsidy, an ERC grant, to look for such hurdles.
But isn’t the arms race between the bacteria and its enemy also a precursor to resistance? If evolution has developed a defense for every phage attack, a phage-based medication seems to be quickly worn out. Browns is not afraid of it. “Those lines have existed for billions of years. Bacteria have been able to build such a memory bank because a few survived an attack and were able to archive it. This application does not change that much. In addition, many disease-causing bacteria do not use the crispr trick. And if a bacteriophage no longer works, you can look for a new one. There are still countless phages in nature that such a bacterium does not know.”
Putting together a superbacteriophage
In turn, the bacteriophages have also learned all kinds of tricks to evade the defenses. “We could collect those tricks and introduce them to existing phages. It is conceivable that we can create a superbacteriophage that does not become resistant so quickly. But to be honest, we still don’t understand the genetic information well enough for that.”
Even bacteria of the same species do not all have the same exterior, explains Brouns. “If you wanted to construct an effective bacteriophage, you would have to know exactly what is important for such a phage. For the time being, we can test this much more quickly on a petri dish. Does one work and the other doesn’t? Then we’ll continue with that one. This also applies to the inside: what kind of defense does the bacterium have, and which phage knows what to do with it and which doesn’t? It is better to test that than to try to predict it.”
About twenty years ago, scientists discovered that some bacteria keep track of who attacked them. The genetic code of these bacteria turned out to contain strange bits of DNA, DNA from a virus. It is a memory bank, says Brouns. This allows the bacteria to act quickly.
“Remember, that cell is packed with thousands of molecules: its own DNA or RNA, nutrients, proteins. That should leave the bacteria undisturbed. With that piece of foreign DNA, he scours his cell, looking for an enemy. If there is a match, he cuts the invaded DNA to smithereens. Some bacteria have stored the DNA of up to a hundred phages in their database.”
Crispr-cas
That genetic memory aid of the bacteria also turns out to be very suitable for tinkering with the DNA of a plant or animal (or human being). Just as the bacterium looks for a match with the DNA of a bacteriophage, the geneticist now uses what has come to be called Crispr-cas to look for a pre-programmed position in the DNA to switch off, correct or replace a gene there.
Brouns played a prominent role in the research into the Crispr-cas technique. It has helped scientists better investigate the function of genes, he says. By switching off a gene, you can see what a cell or tissue can no longer do. This is also useful in research into bacteria and their phages.
Compared to a few years ago, it is already a lot easier to read the genetic code of a bacterium or a phage. Nowadays you are ready for less than a hundred euros. “But then you still don’t understand what it says,” says Brouns. “In that respect, there is still a lot to discover in microbiology. A huge amount of genetic information is stored in databases worldwide. Most of it we don’t understand yet. Or it hasn’t even been looked at yet. What proteins do these genes encode for? What exactly do those proteins do? It is the fruits of billions of years of evolution that are there for the taking. But someone has to see it.”
New breakthroughs
It could just be that this will lead to new breakthroughs. A few years ago, Brouns and his group were digging into such a DNA database. “Then we came across a sequence that we thought: this could be something. Kind of like that Crispr-cas memory bank. Coincidentally, a group from Nijmegen turned out to have the bacteria in question so that we could investigate its function.”
And indeed, it turned out to be a blink. “But it didn’t cut the DNA of a virus like the Crispr-cas, it was a protein cutter. Usually it was ‘off’, but if a suitable piece of virus RNA stuck to it, it started to cut. A protein is a completely different molecule than DNA, much larger and more complex. It is difficult to estimate what we can do with it. But that the blink has potential, everyone in the field agrees. We could well be at the start of a Crispr-like revolution.”
The Ammodo Prices
The Ammodo Awards are presented annually to eight researchers in different disciplines. In even years, the prizes go to team research, while in odd years, the focus is on individual researchers who go beyond the boundaries of current knowledge and insights. This year laureates will each receive 350,000 euros that they can spend on their research as they see fit.
One of the other winners is Tatiana Filatova, who is looking for a model who the impact of climate change on society captures. By combining insights, these models come ever closer to the real world. As her studies of potential flood areas show. Read this interview online at Trouw.nl/ Wetenschap
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