Time to start phages
When antibiotics are powerless
Galina Kostina, "Expert" No. 9-2014
The World Health Organization (WHO) is literally crying out about the catastrophic situation with antibiotics. WHO Head Margaret Chan said at a recent European conference that medicine is returning to the pre-antibiotic era. New drugs are practically not being developed. Resources are exhausted: "The post-antibiotic era really means the end of modern medicine as we know it. Common conditions such as streptococcal sore throat or a scratch on a child's knee can lead to death again." According to WHO, more than 4 million children under the age of five die from infectious diseases every year. The main problem becomes resistance (resistance) bacteria to antibiotics. In Europe, the alarm is sounding: the level of resistance, for example, pneumonia has reached 60% – one and a half times more than four years ago. In recent years, pneumonia and other infections caused only by pathogenic bacteria resistant to existing antibiotics annually claim the lives of approximately 25 thousand Europeans. Many people remember the sensational story in 2011, when more than 2,000 people were infected with acute intestinal infection in Germany, more than 20 people died, and 600 kidneys failed due to the disease. The reason was E. coli E. coli resistant to a number of groups of antibiotics, brought, as first thought, on cucumbers, and then, as it turned out, on fenugreek seedlings. According to WHO forecasts, in ten to twenty years all microbes will become resistant to existing antibiotics.
But nature has a weapon against bacteria. And scientists are trying to put it at the service of medicine.
Bacterial OverseersBacteria have long been considered the largest population of living organisms on Earth.
However, not so long ago it became clear that bacteriophages (bacterial viruses) even more. A little, of course, a strange situation: why then did not the phages destroy all the bacteria? As always, everything is not easy in nature. Nature has arranged the microcosm in such a way that the populations of phages and bacteria are in dynamic equilibrium. This is achieved by the selectivity of phages, the closeness of their communication with the corresponding bacteria, and ways to protect bacteria from phages.
It is believed that phages are almost as ancient as bacteria. They were discovered almost simultaneously by Frederick Twort and Felix D'erel at the beginning of the XX century. The first, however, did not dare to designate them as a new class of viruses. But the second methodically described the viruses of dysentery bacteria and called them bacteriophages – eaters of bacteria in 1917. D'herel, who mixed bacteria and viruses, saw how the culture of bacteria literally dissolved before his eyes. And almost immediately, the French scientist began to make attempts to use viruses against dysentery in a children's clinic. It is curious that then the Frenchman continued his experiments in Tbilisi and opened an institute there that dealt almost exclusively with phage therapy. Following D'erel, many scientists and doctors became interested in phages. Somewhere their experiments were successful and inspiring, somewhere failed. Now it's easy to explain: bacteriophages are very selective, almost every virus opposes a certain bacterium, sometimes even a specific strain of it. Of course, if you treat the patient with the wrong phages, then he will not get better. And in 1929, Alexander Fleming discovered the world's first antibiotic, penicillin, and the era of antibiotics began in the early 1940s. As often happens, bacteriophages were almost forgotten, and only in Russia and Georgia they continued to slowly produce phage preparations.
Interest in bacteriophages was revived in the 1950s, when they began to be used as convenient model organisms. "Many fundamental discoveries in molecular biology related to the genetic code, replication and other cellular mechanisms were made largely thanks to bacteriophages," says Konstantin Miroshnikov, head of the Laboratory of Molecular Bioengineering at the M. M. Shemyakin and Y. A. Ovchinnikov Institute of Bioorganic Chemistry (IBH) of the Russian Academy of Sciences. The explosive development of microbiology and genetics has accumulated vast knowledge about both phages and bacteria.
Vadim Mesyanzhinov's laboratory of the IBH RAS, where Konstantin Miroshnikov, Mikhail Schneider, Peter Leyman and Viktor Kostyuchenko worked together fifteen years ago, dealt with bacteriophages, in particular phage T4. "The so–called tailed phages are divided into three groups," says Miroshnikov. – Some have a small, almost symbolic tail, others have a long and flexible tail, and others have a complex, multicomponent, contractible tail. The last group of phages to which T4 belongs is called miovirids." In the pictures, the T4 resembles a fantastic flying object with a head containing DNA, with a strong tail and legs – sensor proteins. Having found a suitable bacterium with the sensor legs, the bacteriophage attaches to it, after which the outer part of the tail contracts, pushing forward the inner piston piercing the shell of the bacterium. For this, the tail of the phage was nicknamed the molecular syringe. Through the piston, the phage injects its DNA into the bacterium and waits for its offspring to breed in it. After the completion of the reproductive cycle, the phage babies tear the bacterial wall and are capable of infecting other bacteria.
Scientists, according to Konstantin Miroshnikov, did not want to believe for a long time that phage uses such a primitive method – mechanical piercing of bacteria, because almost all biological processes are based on biochemical reactions. Nevertheless, it turned out that it is. However, this is just part of the process. As it turned out later, the outer shell of the bacterium, the plasma membrane, is mechanically pierced. The composition of the molecular syringe contains the enzyme lysozyme, which makes a small hole in the inner shell of the cell. The protein of the "syringe" was of the greatest interest to scientists – its kind of needle that pierces the outer shell. It turned out that, unlike many other proteins, it has a remarkably stable structure, which, apparently, is necessary for such a strong mechanical effect. Russian scientists together with colleagues from Purdue University (USA) have built a molecular model of the T4 phage. Later, while studying the details of this unusual molecular weapon of the bacteriophage, scientists came across another mystery. Electron microscopy performed by Viktor Kostyuchenko showed that there is another small squirrel at the end of the needle. And the laboratory again asked the question: what kind of protein is this and why is it needed? However, at that time it was not possible to understand this. One of Vadim Mesyanzhinov's students, Peter Leyman, who worked after IBH at Purdue University and then at the Swiss Institute of Technology in Lausanne (EPFL), later returned to this topic, however, from the other side – from the bacteria side. One of the focuses of the new laboratory's work is not bacteriophages, but bacteria that attack their unfriendly neighbors with a machine very similar to a molecular phage syringe. In scientific terms, it is called the type 6 secretion system (SS6T). And this system turned out to be even more interesting.
Death at the tip of a needle"The sixth type of secretion system was discovered in 2006," says Peter Leyman.
– However, at that time it was not yet clear how similar it was to the tail of a bacteriophage. This discovery was made thanks to the accumulated knowledge about the sequenced genomes of hundreds of bacteria." Over the next three years of research, it turned out that structurally SS6T is almost the same as the tail of a bacteriophage. It also has an external shrinkable case, an internal piston and a needle with a tip. And this molecular machine punches a hole in the shell of the bacterium. According to Konstantin Miroshnikov, it is quite possible that over millions of years of coexistence, an enterprising bacterium could well adopt its weapon from a bacteriophage in order to use it in the fight against other bacteria. At the same time, the bacterium got rid of the phage "head" – the bacterium did not need someone else's genetic information. But she inserted his wonderful tail into her genome. However, the bacterium has significantly modified it. SS6T is much more complex than a bacteriophage molecular syringe. The bacteriophage makes a neat hole, not intending to instantly kill the bacterium in order to multiply in it later. Bacteria also need to quickly and reliably kill a competitor bacterium, so it immediately makes many large holes in the enemy's body.
Peter Leyman's group, in collaboration with Mikhail Schneider from the IBH laboratory, among other tasks, searched in this system for the very small protein at the end of the syringe that they once saw in bacteriophage T4. They had no doubt that it was there and that it should have an important function in this mechanism. "Many people did not believe that there was something on the tip of the needle and that it could be important," says Peter Leyman. – And we searched hard. And yet we found it!" Scientists have found out that various toxins can attach to this small protein tip, which will inevitably kill another bacterium after it is pierced by the tip. In particular, it turned out that one of these toxins may be lysozyme, an analogue of what sits on the molecular syringe of the phage. But, sitting on the phage, it makes a tiny hole in the cell wall and does not penetrate the bacteria, and in CC6T it destroys the cell wall of the bacterium, which leads to its death. However, lysozyme is not the only toxin that uses bacteria, there are dozens and hundreds of them. Moreover, according to Leyman, they can penetrate into another bacterium, both sitting on the tip and squirting out from inside the syringe. But the tricks don't end there either. It turned out that the bacterium has several such replaceable tips, which it chooses depending on which enemy it is going to attack and what it will regale this enemy with. Well, another innovation of the bacterium: the SS6T system is not disposable, like a molecular syringe of a bacteriophage, but reusable. After it pierces the enemy bacterium and delivers toxins into it, the part of the system that is inside the attacking cell breaks down into elements from which the bacterium collects a new "syringe" - the SS6T system charged with toxins. And ready to fight again.
This is an interesting fundamental discovery (an article dedicated to it was published recently in Nature), however, requires continuation. "So far, one of the most mysterious things for us," Leyman continues, "is how the secretion system selects replacement tips and toxins for transportation. We already have some developments, but we are still in the process." Peter Leyman has no doubt that in the coming years these details will finally be clarified. According to him, several laboratories are working on this only in Switzerland and dozens more laboratories around the world. Knowledge of how the killer mechanism of SS6T works may contribute to the development of a new class of drugs that will selectively kill pathogenic bacteria. Medicine is very much waiting for this discovery.
Time to start phagesThe era of antibiotics, which began in the middle of the last century and caused general euphoria, seems to be ending.
And the father of antibiotics, Fleming, warned about this. He assumed that clever bacteria would invent survival mechanisms all the time. Whenever faced with a new drug, the bacteria seem to pass through the bottleneck. The strongest survive, who have acquired a mechanism of protection against the antibiotic. In addition, the rampant and uncontrolled use of antibiotics, especially in agriculture, hastened the approach of the end of their era. The more actively antibiotics were used, the faster the bacteria adapted to them. Hospital–acquired infections have become a particular problem, the causative agents of which feel at home in the holy of holies - sterile departments of clinics. There, among patients with weakened immunity, even so-called conditionally pathogenic microbes that pose no danger to a healthy person, but have acquired a solid spectrum of antibiotic resistance, become cruel pathogens and finish off patients.
According to Mikhail Schneider, antibiotics, as a rule, are taken from nature, like the same penicillin. There are very few synthesized antibiotics: it is difficult to catch vulnerable places in bacteria that could be targeted. In addition, doctors complain, developers are not very willing to take up the creation of new antibiotics: they say, there is a lot of fuss with developments, resistance to them is produced in bacteria too quickly, and the price for them cannot be as high as, for example, anti-cancer drugs. According to some reports, by the end of the first decade of the XXI century, only a dozen and a half new antibiotics were in the development of large companies, and even then at very early stages. It was then that they began to remember about the natural enemies of bacteria – bacteriophages, which are also good because they are practically non-toxic to the human body.
In Russia, therapeutic phage preparations have been made for a long time. "I was holding in my hands a battered manual from the Finnish War on the use of phages in military medicine, phages were treated even before antibiotics,– says Konstantin Miroshnikov. – In recent years, phages have been widely used in floods in Krymsk and Khabarovsk to prevent dysentery. We have been making such drugs on an industrial scale for many years by the NGO “Microgen". But the technology of their creation has long been in need of modernization. And we have been cooperating with Microgen on this topic for the last three years."
Bacteriophages seem to be a great weapon against bacteria. Firstly, they are highly specific: each phage kills not just its own bacterium, but even its specific strain. According to Mikhail Schneider, bacteriophages could be used both in diagnostic tools to identify bacteria before strains, and in therapy: "They can be used both by themselves and in combination with antibiotics. Antibiotics at least partially weaken the bacteria. And phages can finish them off."
Now many laboratories are thinking about how it would be possible to use both bacteriophages and their components against bacterial infections. "In particular, the American company Avidbiotics develops products based on bacteriocins, which are a modified phage tail – a molecular syringe aimed at destroying harmful bacteria," says Mikhail Schneider. "They have created a kind of molecular constructor that can easily change the sensory protein that recognizes a specific pathogenic bacterium, thanks to which many highly specific drugs can be obtained." Currently, the company is developing drugs that will be directed against E. coli, salmonella, shigella and other bacteria. In addition, the company prepares preparations for food safety and has entered into an agreement with DuPont to create a class of antibacterial agents to protect food.
Russia, it would seem, has a wide road ahead for the creation of new classes of drugs based on phages, but so far there is no energetic action in this regard. "We are not production workers, but we can roughly imagine what a mess the certification and introduction of a modern drug based on phages or bacteriocins can result in," says Miroshnikov. – After all, he will have to go through the path of a new drug, and this takes up to ten years, then it will still be necessary to approve every detail of such a design drug with replaceable particles. So far, we can only give scientific recommendations on what could be done." And there is no doubt in anyone who is aware of the disaster with antibiotics about what needs to be done.
Phages may soon be replaced by new technologies that will use SS6T mechanisms. "We are still in the process of research and are still far from rational use of the secretion system of the sixth type," says Peter Leyman. – But I have no doubt that these mechanisms will be revealed. And then, on their basis, it will be possible to make not only highly specific drugs against malicious bacteria, but also use them as a means of delivering proteins necessary for the body, even very large ones, which is now a problem, as well as delivering drugs, for example, to tumor cells."
Portal "Eternal youth" http://vechnayamolodost.ru24.02.2014