An alternative to antibiotics
Antimicrobial peptides
Alexey Vasilchenko, Candidate of Biological Sciences, Institute of Ecological and Agricultural Biology (X-BIO), Tyumen State University.
Antimicrobial peptides are protective molecules that provide innate immunity to almost all living beings. From the point of view of chemistry, peptides are a sequence of amino acids of a certain length in a chain. This is a diverse group of molecules of natural or synthetic origin that have antimicrobial properties. They are able to interact with bacterial cells and act either bactericidal, that is, kill them, or bacteriostatically – slow down their physiology, which is why the cells stop dividing and growing.
Antimicrobial peptides are produced by almost all living organisms. This is an evolutionarily ancient way of protecting organisms from the surrounding effects of pathogenic bacteria, a means of innate immunity: starting from the embryo, these molecules protect the body. Vertebrate organisms have acquired immunity – antibodies that are produced if the body gets over an infection, and there are innate antimicrobial peptides: the body comes with a means of protection against bacteria, viruses and parasites.
Classification of antimicrobial peptides
Antimicrobial peptides are difficult to classify based on their structural diversity. The Antimicrobial Peptides database (as of October 2018) contains information about three thousand peptides. It includes peptides of animals, plants and bacteria.
There are two ways of natural peptide synthesis: ribosomal synthesis and non-ribosomal synthesis. Peptides synthesized by ribosomes are produced by almost all organisms. Their classification is based on the secondary structure that the molecules form in aqueous solutions. There are α-helical peptides, peptides with a β-folded structure and with an unordered (random) structure.
Antimicrobial peptides synthesized by microorganisms are even more diverse: microorganisms are able not only to synthesize non-ribosomally, but also then modify the molecules synthesized on ribosomes. Posttranslational modifications give peptides additional properties. For example, they recognize targets better and are more stable, and therefore more functional compared to ribosomally synthesized animal peptides.
Peptides as an alternative to classical antibiotics
Antimicrobial peptides have been considered for decades as an alternative to conventional antibiotics that are used in the treatment of infectious diseases. Antibiotics are now no longer working, in some cases only "last hope" antibiotics help: when the choice is between whether a patient will die from sepsis or his kidneys will refuse antibiotics, antibiotics of last hope are prescribed.
Antimicrobial peptides have a unique ability to overcome the virulence and resistance of bacteria by affecting the most conservative structures of the microbial cell. Antimicrobial peptides are less toxic than classical antibiotics and decompose in the human body very quickly. But this is also connected with the fact why peptides are still not widely used: they are excreted from the body too quickly. It is ineffective to inject them intravenously: peptides will not go far through the bloodstream. And it is pointless to prescribe them through the gastrointestinal tract, because they will split in the stomach. At the moment, there are not so many approved and clinically tested drugs based on antimicrobial peptides, and they are very expensive.
Antimicrobial peptides are used not only in medicine. For example, the antimicrobial peptide of bacterial origin nizin has been used in the food industry for more than 50 years. In the composition of the products, you can find the additive E-234 – this is nizin. This peptide acts on pathogenic flora that causes spoilage of products, for example on listeria – these are pathogens of deadly diseases of animals and humans, which are transmitted mainly with food.
Another positive property of antimicrobial peptides and the reason why they are not excluded as an alternative to classical antibiotics is that peptides act on bacteria, inhibit them, while resistance to them is produced, but not as often as in the case of conventional antibiotics.
Antibiotic resistance
Bacteria form resistance to classical antibiotics due to gene transfer. The bacterium is able to replace one amino acid with another in the ribosome, and the antibiotics associated with the ribosome cease to work. Antimicrobial peptides act on bacterial cell structures that are very difficult for bacteria to replace. For example, peptides act on the bacterial wall, and no bacteria can completely change the structure of the wall.
Antimicrobial peptides are introduced into the cell wall and create pores in it, from which the intracellular contents of the bacterium flow out. In some cases, the membrane is torn to pieces, and the cell falls apart. Peptides work very quickly: if a conventional antibiotic takes hours to act, then the peptide destroys the bacterial cell in a matter of seconds. The faster the drug acts on the cell, the less likely it is that the cell will begin to divide and form stable forms.
Resistance to antimicrobial peptides also occurs, but much less frequently than to conventional antibiotics.
Sub-inhibitory effect
Under natural conditions, and not in a test tube, the concentration of peptides necessary for the destruction of microorganisms is not always achievable for various reasons, including elementary dilution in the surrounding moisture. What happens if you don't find a sufficient concentration that will kill cells? What will happen to a bacterial cell affected by a low concentration of the substance? The effects can be different – both negative and positive for a person.
The cell may react in such a way that it becomes more aggressive and turns into a "super monster", or it may have a positive effect: the cell will stop releasing toxins, but it will not die. What does this or that scenario depend on?
Peptide molecules are usually positively charged, so they interact electrostatically with the bacterial cell wall, which is negatively charged. Membrane structures that separate cellular contents from the environment have many sensors: both human nerve endings on the skin and bacteria have sensors in membrane structures. Such a system consists of a sensory protein (histidine kinase) and a corresponding response regulator. Sensory kinase is attached from the inside to the bacterial cytoplasmic membrane and has a sensitive tail on its outer side. Most antimicrobial peptides are positively charged, and this allows them to interact directly with this part of the sensor, which leads to the activation of the signaling system.
If the cell did not die immediately when exposed to peptides, the sensors interact with the peptides and begin to transmit a signal to the gene apparatus that responds to these specific signals. For example, it can change the cell walls in the outer membrane: replace one molecule with another and increase the charge. The peptide is positively charged, the cell wall is negatively charged, so you need to make the membrane charge positive, and similarly charged molecules will repel like magnets. "Pumps" can start working, which will pump peptides out of the cell, or products will be released from proteolytic enzymes and break down peptides threatening the cell.
Bacterial membrane sensors react to some peptides, but not to others. There is a suspicion that there is a certain specificity and cells can recognize which peptide is in front of them. There is no clear mathematical statistical description of this process yet, and it is impossible to say with certainty whether there will be a reaction to a specific molecule or not.
The sub–inhibitory effect is relatively better studied in the case of conventional antibiotics, which are available in the form of pharmaceuticals - their effect in sub-inhibitory concentrations has been studied for a long time. It is already well known that small concentrations of antibiotics can cause unexpected reactions from the bacterial population. For example, fluoroquinolones can stimulate bacterial adaptation to various stresses, including the action of antibiotics themselves, and penicillins can enhance the formation of biofilms.
Peptides of a wide and narrow spectrum of action
Antimicrobial peptides of animals have a wide spectrum of action: peptides in the skin secretions of the spur frog act on almost all types of bacteria – both gram-positive and gram-negative.
Gram-positive and gram-negative bacteria differ in the structure of the cell wall: due to a more powerful and impenetrable cell wall, gram-negative bacteria are more resistant than gram-positive ones. For example, the antibiotic penicillin slows down the growth of the cell wall and acts better on gram-positive bacteria than on gram-negative ones. In addition, there are cells that do not have an outer membrane, and there are cells that have an outer membrane that acts as an additional barrier against peptides – broad-spectrum antimicrobial peptides act on all these cell types.
Bacteriocins are antimicrobial peptides produced by bacteria. They are distinguished from the peptides produced by the frog by the fact that bacterial peptides act only on representatives of their kind: peptides produced by enterococcus will destroy only enterococci.
When conducting therapy, it is necessary to act selectively on a certain group of bacteria so that the entire microflora does not die in a person. To do this, it is possible to create combined preparations based on peptides of a narrow and wide spectrum of action.
Preparation of peptides
Antimicrobial peptides are obtained by chemical synthesis, but there are limitations associated with the length of the peptide: synthesizing a peptide of more than 30 amino acid residues is quite difficult. Chemically synthesized peptides cannot be placed in space in the necessary structure. Peptides synthesized by a living organism have a certain clutch, which plays a role in antimicrobial properties, and the synthesized molecule will be inferior to its natural counterpart.
You can do drag design and replace one amino acid in a molecule with another. At the same time, the physicochemical properties of the peptide change: scientists achieve greater antimicrobial activity - less substance is required to kill cells – and less cytotoxicity – the peptide kills only bacterial cells and does not interact with red blood cells. It is possible to achieve greater resistance to the action of proteolytic enzymes of the body that destroy peptides, so that oral administration of peptides can be used without fear of their destruction in the gastrointestinal tract. With the help of chemistry methods, you can play with natural molecules and improve their properties. This approach also has its advantages: we cannot synthesize the molecule that we would like, but we can modify it and change its properties.
If you know the sequence of amino acids, you can construct a plasmid: translate the sequence of nucleotides into a mini-gene and embed it, for example, into E. coli, and E. coli as a reactor will synthesize peptides. This is a genetically engineered way of obtaining substances - this is how insulin and many other natural medicines are obtained.
Portal "Eternal youth" http://vechnayamolodost.ru