Humanity can win the War against Bacteria

Pavel Nazarov, Candidate of Biological Sciences, Research Institute of Physico-Chemical Biology, Moscow State University
Kommersant Nauka No. 5, July 2017
Published on the website "Elements"

In May of this year, in the work "Mitochondria-targeted antioxidants as highly effective antibiotics", published in the journal Scientific Reports, a team of authors from Moscow State University for the first time showed a fundamentally new hybrid antibiotic: its action is directed against the membrane potential of bacteria, which provides pathogenic cells with energy.

Victory! – but only temporary

In the middle of the last century, humanity was in a state of euphoria associated with incredible success in the treatment of infectious diseases of bacterial nature. Many bacterial infections that caused horrific epidemics in the Middle Ages in terms of the number of victims turned into quarantine infections that were easily and effectively cured.

This success became possible after the discovery in the 1920s by the British bacteriologist Alexander Fleming of the first antibiotic - penicillin; it was found in mold fungi Penicillium notatum. A decade later , British scientists Howard Florey and Ernst Chain proposed a method for the industrial production of pure penicillin. All three were awarded the Nobel Prize in Physiology or Medicine in 1945.

Mass production of penicillin was established during the Second World War, which caused a sharp decrease in mortality among soldiers who usually died from wound infections. This allowed French newspapers to write on the eve of Fleming's visit to Paris that he had made more entire divisions to defeat fascism and liberate France.

The deepening of knowledge about bacteria has led to the emergence of a large number of antibiotics, diverse in mechanism, spectrum of action and chemical properties. Almost all bacterial diseases were either completely cured or seriously suppressed by antibiotics. People believed that man had defeated bacterial infections.

Small pockets of resistance – and defeat

Simultaneously with the successes, the first signs of an impending global problem appeared: cases of bacterial resistance to antibiotics. Previously, microorganisms that were uncomplainingly sensitive to them suddenly became indifferent. Humanity responded with a rapid development of research and new antibiotics, which only led to an increase in the number of drugs and a new resistance of bacteria.

In May 2015, the World Health Organization recognized bacterial resistance to antibiotics as a crisis and put forward a Global Plan to Combat Antimicrobial Resistance. It had to be carried out without delay, numerous international organizations such as environmentalists and economic sectors had to coordinate their actions – not only human medicine, but also veterinary medicine, industrial animal husbandry, financial institutions, and consumer protection societies.

The plan must be being implemented one way or another, but unfortunately, despite this, already in September 2016, one American patient died of sepsis. This happens, and even more often than we would like, but it was destroyed by the so–called superbug - Klebsiella pneumoniae, but not ordinary, but resistant to all 26 antibiotics allowed in the USA, including the "last reserve" antibiotic colistin.

The last frontier has fallen

Colistin is considered an antibiotic of the last reserve – it is an old drug from the class of polymyxins that has fallen out of use due to its toxic effect on the kidneys. When superbugs were discovered, which, in addition to resisting known antibiotics themselves, also acquired the ability to transmit gene information to each other that allows them to resist antibiotics, it turned out that, firstly, colistin is harmful to all these bacteria, and secondly, bacteria cannot exchange colistin resistance genes, if suddenly all- it will still arise.

Alas, but in May 2016, the American Repository of multi-resistant microorganisms, which is located in the structure of the Walter Reed Research Institute (this is the structure of the US Army), received a bacterium that was not just indifferent to colistin, but also turned out to be able to transmit gene information with this resistance to other bacteria. The first such microorganism was recorded in China back in 2015, for a long time there was hope that this was an isolated case, but it was not justified. It is especially sad that in the USA this microorganism turned out to be a well-known E. coli./NOTE

So, it became obvious to scientists that bacterial infections are defeating humanity, and modern medicine can be discarded in the days before the discovery of antibiotics. One of the main issues raised at the ASM Microbe International Conference, held in In New Orleans in June 2017, the American Society of Microbiologists, was like this: "Can humanity win the war with microbes?". At the same conference, by the way, special attention was paid to the antimicrobial stewardship movement, or the management of antibiotic therapy, which aims to prescribe antibiotics as reasonably and sufficiently as possible, in accordance with the recommendations of evidence-based medicine. So far, such treatment of antibiotics has become law only in one place in the world – in the state of California, USA.

Antioxidants are sent to the mitochondria

But the solution bypassing the resistance of bacteria can be considered to have been found – by Russian scientists. In May of this year, in the work "Mitochondria-targeted antioxidants as highly effective antibiotics", published in the journal Scientific Reports, a team of authors from Moscow State University for the first time showed a fundamentally new hybrid broad–spectrum antibiotic - a mitochondrially directed antioxidant.

Mitochondrially directed antioxidants (MNA) have become widely used not only as a tool for studying the role of mitochondria in various physiological processes, but also as therapeutic agents. These are conjugates, that is, compounds consisting of some well-known antioxidant (plastoquinone, ubiquinone, vitamin E, resveratrol) and penetrating, that is, able to overcome the cell membrane or mitochondria, cation (triphenylphosphonium, rhodamine, etc.).

The mechanism of action of MNA is not known for certain. It is only known that in mitochondria they partially dissociate oxidative phosphorylation, the metabolic pathway of synthesis of a universal cellular fuel – adenosine triphosphate, ATP, which stimulates cellular respiration and reduces the membrane potential and can lead to a protective effect under oxidative stress.

Presumably it looks like this. Due to their lipophilicity (craving for lipids or affinity with them), MNAs bind to the mitochondrial membrane and gradually migrate into the mitochondria, where they apparently combine with a negatively charged fatty acid residue; after forming a complex, they lose charge and again find themselves outside the mitochondrial membrane. There, the fatty acid residue captures a proton, which causes the complex to disintegrate. The fatty acid that has captured the proton is transferred in the opposite direction – and loses a proton inside the mitochondria, that is, in other words, transfers it to the mitochondria, which is why the membrane potential decreases.

One of the first MNAs was created on the basis of triphenylphosphonium in Oxford by the English biologist Michael Murphy; it was a conjugate with ubiquinone (or coenzyme Q, which participates in oxidative phosphorylation). Under the name MitoQ, this antioxidant has gained considerable fame as a promising drug for slowing down skin aging, as well as as a possible means of protecting the liver in hepatitis and its fatty degeneration.

Later, the group of Academician Vladimir Skulachev from Moscow State University followed the same path: an effective SkQ1 was created on the basis of triphenylphosphonium conjugate with the antioxidant plastoquinone (involved in photosynthesis).

According to the symbiotic theory of the origin of mitochondria, put forward by corresponding member of the USSR Academy of Sciences Boris Mikhailovich Kozo-Polyansky in the 1920s and American biologist Lynn Margulis in the 1960s, there are many similarities between mitochondria and bacteria, and it can be expected that MNAs will affect bacteria. However, despite the obvious similarity of bacteria and mitochondria and ten years of experience with MNA all over the world, no attempts to detect the antimicrobial effect of MNA have led to positive results.

The Riddle of two sticks

The breakthrough happened in 2015: for the first time, the antibacterial effect of MNA on the example of SkQ1 was shown in the work "The uncoupling and toxic effect of alkyl-triphenylphosphonium cations on mitochondria and bacteria Bacillus subtilis depending on the length of the alkyl fragment" – it was published by the journal "Biochemistry" in December 2015. But that was a description of the phenomenon: the effect was observed when working with hay bacillus (Bacillus subtilis) and was not observed when working with E. coli (Escherichia coli).

But further research, which formed the basis of the latest work published in the journal Scientific Reports, showed that MNA SkQ1 is a highly effective antibacterial agent against a wide range of gram–positive bacteria. SkQ1 effectively inhibits the growth of annoying bacteria such as Staphylococcus aureus – one of the four most common types of microorganisms that cause nosocomial infections. SkQ1 also effectively suppresses the growth of mycobacteria, including Koch's bacillus (Mycobacterium tuberculosis). Moreover, MNA SkQ1 has proven to be highly effective against Gram-negative bacteria such as Photobacterium phosphoreum and Rhodobacter sphaeroides.

And only with regard to E. coli, it was extremely ineffective, and it was Escherichia coli that is the bacterium that microbiologists use as a model organism, which was, apparently, the reason for unsuccessful attempts to detect the antimicrobial effect of MNA earlier.

Naturally, the exceptional resistance of E. coli caused a very strong interest of researchers. Fortunately, modern microbiology has made a big step forward in the methodological aspect, and scientists have created entire collections of microorganisms with deletions (absence) of some genes that do not cause their death. One of such collections – deletion mutants of E. coli – is at the disposal of MSU.

The researchers suggested that resistance may be due to the work of any of the pumps of multidrug resistance available in E. coli. Any pump is bad for an infected person because it simply throws an antibiotic out of a bacterial cell, it does not have time to act on it.

How the pump works

The action of the pump can be illustrated by the example of the main pump of multidrug resistance of E. coli – AcrAB-TolC. 

This pump consists of three main components: (1) protein of the inner cell membrane AcrB, which, due to the membrane potential, can move substances through the inner membrane (2) of the AcrA adapter protein binding the AcrB transporter to (3) a channel on the outer TolC membrane. 

The exact mechanism of the pump operation remains insufficiently studied, however, it is known that the substance that the pump should throw out of the cell gets to the inner membrane, where the AcrB transporter is waiting for it, binds to the active center of the pump and then, due to the energy of the oncoming proton movement, is pumped out of the outer membrane of the bacterium.

E. coli has a lot of genes responsible for the action of multidrug–resistant pumps, and it was decided to start the analysis with the products of genes that are part of several pumps at once, namely the TolC protein.

The TolC protein is a channel on the outer membrane of gram–negative bacteria, it serves as the outer part for several pumps of multidrug resistance.

Analysis of the deletion mutant (i.e., a rod without TolC protein) showed that its resistance decreased by two orders of magnitude and became indistinguishable from the resistance of gram-positive bacteria and non-resistant gram-negative bacteria. Thus, it could be concluded that the outstanding resistance of E. coli is the result of the work of one of the multidrug–resistant pumps containing TolC protein. And further analysis of deletion mutants by protein components of multidrug–resistant pumps showed that only the AcrAB-TolC pump is involved in pumping out SkQ1.

Resistance caused by the presence of the AcrAB-TolC pump does not look like an insurmountable obstacle: the antioxidant conjugate SkQ1 is also a unique substance for this pump, obviously, it will be possible to find an inhibitor for it.

Not only to treat, but also to repair

But to be called an antibiotic, SkQ1 must meet a variety of criteria, such as (1) the ability to suppress the vital processes of microorganisms in small concentrations and (2) little or no damage to human and animal cells. Comparison of SkQ1 with known antibiotics – kanamycin, chloramphenicol, ampicillin, ciprofloxacin, vancomycin, etc. – showed that SkQ1 acts on bacteria in the same or even lower concentrations. Moreover, in a comparative study of the effect of SkQ1 on human cell culture of the HeLa line, it turned out that in the minimum bactericidal concentration, SkQ1 has virtually no effect on human cells – and SkQ1 cells are noticed when the concentration of the antioxidant conjugate becomes more than an order of magnitude higher than necessary for bactericidal action.

The mechanism of action of SkQ1 on bacteria turned out to be similar to the action of MNA on mitochondria, but the overall effect on prokaryotic and eukaryotic cells differed. One of the main reasons is the spatial separation of energy generation processes (excluding substrate phosphorylation) and the processes of transport of substances into the cell, which, apparently, represents a significant evolutionary advantage that is often overlooked when considering the benefits of cohabitation of protomitochondria and proto–eukaryotes. Since energy generation and transport in bacteria are localized on the cell membrane, the drop in potential apparently causes both processes to stop at once, which leads to the death of the microorganism. In the eukaryotic cell, the processes of transport of substances into the cell are localized on the cell membrane, and energy generation occurs in the mitochondria, which allows the eukaryotic cell to survive at lethal concentrations of MNA for bacteria. In addition, the potential difference on the membrane of a bacterium and a eukaryotic cell differs in favor of bacteria – and this is the same additional factor accumulating MNA on the membrane of bacteria.

Considering the mechanism of action of SkQ1 on bacteria, it is impossible to ignore another unique property of this MN – the ability to treat eukaryotic cells damaged by bacteria due to antioxidant properties. SkQ1, acting as an antioxidant, reduces the level of harmful reactive oxygen species formed during inflammation caused by bacterial infection.

Thus, SkQ1 can be recognized as a unique hybrid antibiotic of the widest spectrum of action. Further development of antibiotics based on it may allow to reverse the course of the war of mankind against increasingly advanced microbes.

Portal "Eternal youth" http://vechnayamolodost.ru  31.08.2017