23 March 2017

In search of antibiotics

About the discovery of penicillin, uncultivated forms of bacteria and antibiotics acting on protein biosynthesis

sergiev.jpgPeter Sergiev, Post-science

Many have heard the story of the discovery of the first antibiotic – penicillin – by the British scientist Alexander Fleming. This discovery was largely luck, and in many ways it was due to Fleming's observation. He was growing colonies of bacteria on agar cups, and a mold spore accidentally flew into one of these cups. Another person might have thrown out such a cup, but Fleming noticed that bacteria were lysing around the mold colony, that is, they died. He isolated active compounds from this mold, which were later called penicillin, and penicillin derivatives are still in clinical practice.

The first synthetic antibiotic was discovered by German researcher Gerhard Domagk in 1932. He examined the dyes and found that sulfanilamide and its derivatives have an antibacterial effect. These compounds are still used in clinical practice.

The first Soviet antibiotic, gramicidin C, was discovered during the Great Patriotic War. At that time there were many wounded, wounds often rotted, and antibiotics were needed against festering wounds. Accordingly, a program to search for antibiotics began in our country, and Georgy Frantsevich Gause, together with Maria Georgievna Brazhnikova, under the leadership of Pyotr Grigorievich Sergiev, opened the first Soviet antibiotic. In the future, the school of Gause and Brazhnikova discovered many antibiotics that found their use in the clinic. Now the Gause Institute is still actively working in Russia and is looking for new antibiotics.

Most antibiotics were discovered in the 50-60s of the XX century, and it was the golden age of antibiotics. They were opened in many, in all countries of the world. We searched for them in this way: individual colonies of soil microorganisms, fungi or bacteria were isolated from natural habitats, for example, from the soil, we looked at whether these colonies could kill bacteria around them, we isolated an active compound. It was a rather laborious process: it was necessary to build up a sufficient amount of producer. It is quite difficult to clean the active compound, and then it is no less difficult to identify its structure.

At that moment it seemed that the problem of bacterial infections had been solved and antibiotics would always protect us. Unfortunately, it turned out that, rather, humanity received a reprieve for several decades in the fight against bacterial infections. The fact is that when you isolate a new producer from some natural environment, a new bacterium that produces an antibiotic, then you don't know what kind of antibiotic it does. You have to isolate it in its pure form, determine the structure, and then you can realize with horror that you have isolated something already known. And it began to turn out more and more often that researchers isolated the same antibiotics repeatedly, and new antibiotics began to occur less and less often. It got to the point that new antibiotics began to be discovered every few years, and eventually once every decade.

To this was added a significant loss of interest in the development of antibiotics from pharmaceutical companies. The fact is that, firstly, the effectiveness of this process decreased, and secondly, it turned out that not so much money can be saved for antibiotics, because a person, one way or another, takes an antibiotic for a fairly short time - one or two weeks. This cannot be compared with medicines against chronic diseases that a person is forced to take all his life.

To reduce the effectiveness in the search for new antibiotics, the spread of pathogenic bacteria that are resistant to antibiotics has been added. The thing is that if you use an antibiotic, and even if there were initially very few resistant bacteria in the bacterial population, then after killing all sensitive bacteria with an antibiotic, that small proportion of resistant ones gets all the resources, and they begin to multiply avalanche-like. Now, pathogenic bacteria that are resistant to them are known for almost all antibiotics that are used in clinical practice. Moreover, there are bacteria that are resistant to several, even most of the antibiotics used, so although we may still feel protected to some extent now, in the future the nightmares of epidemics of bacterial infections may return, so antibiotics should be sought.

I have already described the first method of finding antibiotics to you. This is a classic way of isolating individual colonies of producers from the natural environment, their cultivation. What can be used to increase the efficiency of this process? One of the alternatives is testing chemical compounds that are synthesized in the laboratory. At the moment there are quite extensive collections of chemical compounds – hundreds of thousands and millions of different substances. The advantage in screening among chemical compounds is that we know in advance what we are testing. In other words, there is no problem identifying the compound, there is no problem isolating the compound, there is no problem rediscovering a previously known antibiotic.

The main drawback is due to the fact that natural antibiotics have been perfected by microorganisms for many millions of years, they have achieved great efficiency in killing bacteria. Compounds randomly selected, as a rule, do not have antibacterial activity or have rather weak antibacterial activity. Nevertheless, there is still such a method, and in part we use it in our work. The fact is that even if a randomly found compound weakly kills bacteria, it can be improved ― either blindly, making some random modifications of the original compound, or in a directed way: if we know the target, if we know how this compound works, we can figure out how to improve it.

The next option for improving the search for antibiotics is associated with uncultivated bacteria. The fact is that not all bacteria in nature can be grown on agar in the form of individual colonies. Moreover, it is believed that most bacteria cannot be grown like this. Why is this happening? Why don't bacteria want to grow separately? In a bacterial community, bacteria can not only harm each other, but also help each other. Bacteria of different species can exchange the chemical compounds they need, and thus a certain division of labor in chemical synthesis between bacteria is obtained. As an example, not all bacteria are able to extract iron. We are talking, of course, about iron ions, which bacteria need. Some species are able to release substances into the environment that capture iron ions, and they can be used by their neighbors. This is not the only example of cooperation between bacteria. Uncultivated bacteria can also produce antibiotics. What about them?

Our former compatriot, and now an outstanding American scientist Kim Lewis has come up with a way to cultivate uncultivated bacteria, no matter how strange it may sound. To do this, he uses microscopic containers with semi-permeable walls. One bacterial cell gets into each such container, the bacterium cannot escape from this container, it sits as if in a small prison. But because the walls are permeable, it can exchange substances with other bacteria and the environment. Bacteria enclosed in such cells are placed in their natural habitat, and there they can grow well. Thus, Kim Lewis found a producer of a new antibiotic that acts on the synthesis of the bacterial cell wall.

Another way to find new antibiotics is used by Konstantin Severinov from Skoltech. He is looking for genes that are responsible for the biosynthesis of antibiotics. In order to search for genes, it is absolutely not necessary to grow bacteria ― you can simply determine the nucleotide sequences of DNA, including uncultivated bacteria or bacteria that are poorly cultivated. You can search for genes simply in a computer, in databases on known bacterial genomes. And then such genes can be planted in a well-known bacterium, which is easy to work with, and force it to make antibiotics. This method also has its drawbacks, because the genes may not work in a foreign bacterium.

Our group is studying antibiotics that act on protein biosynthesis, on the ribosome. We are just starting this work at Skoltech, so all the work is based on the foundation that was laid at Moscow State University, at the scientific school of Academician Alexey Alekseevich Bogdanov under the leadership of Olga Anatolyevna Dontsova. An outstanding young man in our group, Ilya Osterman, came up with the idea of making a so-called reporter bacterium. What does it mean? When a bacterium dies from an antibiotic, we don't know which system is broken in it. She's just dying quietly and doesn't show us why in any way. The idea was that the bacterium, when some system stops working for it, begins to glow with a different color, that is, to fluoresce, depending on which mechanism it has broken. So far, we have taught the bacterium to signal to us that, for example, protein biosynthesis is disrupted or DNA begins to be damaged, as, for example, happens when its DNA gyrase is damaged. Using this method, we screen both chemical libraries and libraries of natural compounds and have already found several interesting promising molecules that we are currently studying and trying to improve.

About the author:
Peter Sergiev – Associate Professor, Center for Data-Intensive Biomedicine and Biotechnology, Scoltech.

Portal "Eternal youth" http://vechnayamolodost.ru  23.03.2017

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