Practical application of phages. Bacteriophages: modern aspects of application, prospects for the future

Distinctive properties of bacteriophages as representatives of the Vira kingdom. Virulent phages, stages of interaction with a bacterial cell. Practical use bacteriophages

Virulent phages cause productive infection, at which reproduction of phages and lysis of a bacterial cell occurs.

The mechanism of interaction of a virulent phage with a microbial cell:

1. Phage adsorption on sensitive cells. Occurs in the presence of complementary receptors in the cell wall of bacteria and at the ends of the filaments of the phage process. First, the phage is attached by threads, and then firmly attached to the cell wall with the help of the teeth of the banal plate.

2. Entry of phage DNA into a bacterial cell. With the help of lysozyme, located in the banal plate, a section of the cell wall is hydrolyzed, the sheath of the process is reduced and the inner rod is pierced by the cell membrane. The phage DNA molecule enters the cell through the channel of the rod.

3. Intracellular phage development. Phase DNA brings genetic information into the bacterial cell. There is a biosynthesis of components necessary for reproduction. On the early stages"early proteins" are synthesized - enzymes that replicate phage DNA in order to form many copies of it. Then on cellular ribosomes structural "late proteins" are formed

4. Phage morphogenesis. Phage maturation occurs along three independent branches in different parts of the cell, which is a dissociated process. Separately, phage heads are formed - a capsid is built around the DNA molecule. Independently, the process is being built. Process filaments are synthesized separately. Then all the constituent parts of the phage are combined to form virions.

5. Lysis of the bacterial cell and release of the phage. Lysis is carried out under the action of lysozyme. Exit by budding.

The strict specificity of bacteriophages allows them to be used for phage typing and differentiation of bacterial cultures, as well as for their indication in the external environment, for example, in water bodies.

The method of phage typing of bacteria is widely used in microbiological practice. It allows not only to determine the species affiliation of the culture under study, but also its phagotype (fagovar). This is due to the fact that bacteria of the same species have receptors that adsorb strictly defined phages, which then cause their lysis. The use of sets of such type-specific phages allows phage typing of the studied cultures for the purpose of epidemiological analysis of infectious diseases: (determining the source of infection and ways of its transmission)

II. Phages are used to prevent and treat infectious diseases:

a) phage prophylaxis- a method of preventing the development of certain bacterial infections by ingestion of a specific bacteriophage. Used to prevent cholera, dysentery, typhoid fever, etc.

b) phage therapy is a method of treating bacterial infections by ingesting a specific phage.(typhoid, salmonella, dysentery, proteus, pseudomonas, staphylococcal, streptococcal, coli-phage and combined drugs. They are used in the treatment of infectious diseases caused by the above microorganisms, as well as in the treatment of wound and anaerobic infections.)

Genotypic variability

Pathogenicity -

Adhesion

Invasion

Aggression.

4. Structure of the genetic apparatus of prokaryotes. Phenotypic and genotypic variability. Genetic bases of bacterial pathogenicity.

The genetic apparatus of prokaryotes- does not have a nuclear envelope and is represented by one circular DNA molecule, which is a chromosome; located in the cytoplasm, does not contain histone proteins. Not capable of mitosis

Phenotypic variability - modifications (changes in more than one or more traits) - does not affect the genotype. Phenotypic changes occur under the influence of environmental factors. Modifications affect the majority of individuals in the population. They are not inherited and fade over time, i.e., return to the original phenotype.

Genotypic variability- changing the properties of bacteria, affecting their genotype. It is inherited, it is long-term. Occurs as a result of mutations or genetic exchange (transformation, conjugation or transduction)

Pathogenicity - a species trait that is inherited, fixed in the genome of a microorganism, i.e. it is a genotypic trait that reflects the potential ability of a microorganism to penetrate into a macroorganism and multiply in it (invasiveness), cause a complex pathological processes that occur during illness.

Pathogenicity factors include the ability of microorganisms to attach to cells (adhesion), settle on their surface (colonization), penetrate cells (invasion), and resist the body's defense factors (aggression).

Some of them are encoded directly by the nucleoid genes (for example, the capsule and enzymes in some species). The other part is encoded by extrachromosomal factors of heredity - plasmids and episomes. Plasmid genes usually determine the interaction of pathogens with the epithelium, while chromosomal genes determine the existence and reproduction of bacteria extracellularly in organs and tissues.

Adhesion The structures responsible for binding a microorganism to a cell are called adhesins and they are located on its surface. In gram-negative bacteria, adhesion occurs due to pili I and general types. In Gram-positive bacteria, adhesins are proteins and teichoic acids of the cell wall. In other microorganisms, this function is performed by various structures of the cellular system: surface proteins, lipopolysaccharides, etc.

Invasion the enzyme hyaluronidase breaks down hyaluronic acid, which is part of the intercellular substance, and thus increases the permeability of the mucous membranes and connective tissue. Neuraminidase breaks down neuraminic acid, which is part of the surface receptors of mucous membrane cells, which contributes to the penetration of the pathogen into tissues.

Aggression Aggression factors include: proteases - enzymes that destroy immunoglobulins; coagulase - an enzyme that coagulates blood plasma; fibrinolysin - dissolving fibrin clot; lecithinase - an enzyme that acts on the phospholipids of the membranes of muscle fibers, erythrocytes and other cells .

Practical application of phages. Bacteriophages are used in the laboratory diagnosis of infections during the intraspecific identification of bacteria, i.e., the determination of the phagovar (phage type). To do this, the phage typing method is used, based on the strict specificity of the action of phages: drops of various diagnostic type-specific phages are applied to a cup with a dense nutrient medium seeded with a "lawn" of a pure culture of the pathogen. The phage phage of a bacterium is determined by the type of phage that caused its lysis (the formation of a sterile spot, "plaque", or "negative colony", phage). The phage typing technique is used to identify the source and ways of spreading the infection (epidemiological marking). Isolation of bacteria of the same fagovar from different patients indicates a common source of their infection.

Phages are also used to treat and prevent a number of bacterial infections. They produce typhoid, salmonella, dysentery, pseudomonas, staphylococcal, streptococcal phages and combined preparations(coliproteic, pyobacteriophages, etc.). Bacteriophages are prescribed according to indications orally, parenterally or topically in the form of liquid, tablet forms, suppositories or aerosols.

Bacteriophages are widely used in genetic engineering and biotechnology as vectors for obtaining recombinant DNA.

Used in practice, bacteriophage preparations are a filtrate of a broth culture of the corresponding microbes lysed by a phage, containing living phage particles, as well as dissolved bacterial antigens released from bacterial cells during their lysis. The resulting preparation - a liquid bacteriophage should look like a completely transparent yellow liquid of greater or lesser intensity.

For therapeutic and prophylactic purposes, phages can be produced in the form of tablets with an acid-resistant coating. Tableted dry phage is more stable during storage and convenient to use. One tablet of dry bacteriophage corresponds to 20-25 ml liquid preparation. The shelf life of the dry and liquid preparation is 1 year. Liquid bacteriophage should be stored at a temperature of + 2 +10 C, dry - no higher than +1 ° C, but it can be stored in a refrigerator at a negative temperature.

The bacteriophage taken orally remains in the body for 5-7 days. As a rule, taking a bacteriophage is not accompanied by any reactions or complications. There are no contraindications for admission. They are used in the form of irrigations, rinses, lotions, tampons, injections, and also injected into the cavities - abdominal, pleural, articular and bladder, depending on the location of the pathogen.

Diagnostic phages are produced both in liquid and dry form in ampoules. Before starting work, the dry bacteriophage is diluted. If the titer, tr, DRT (working titer dose) is indicated on the ampoules, it is used in the phage lizability test (Otto method) to identify bacteria, if the phage type is indicated, then for phage typing - to determine the source of infection.

The action of a bacteriophage on a microbial culture in a liquid medium and on a dense medium

Otto's Method (Falling Drop)

Make a dense sowing lawn of the crop under study. 5-10 minutes after sowing, a liquid diagnostic phage is applied to the dried surface of the nutrient medium. The dish is tilted slightly so that the drop of phage spreads over the surface of the agar. The cup is placed in a thermostat for 18-24 hours. The result is accounted for total absence culture growth at the site of phage droplet application.

Experiment on a liquid nutrient medium

Do sowing of the studied culture in two test tubes with a liquid medium. A diagnostic bacteriophage is added in a loop into one test tube (“O”). After 18-20 hours in a test tube where the bacteriophage was not added (“K”), a strong clouding of the broth is observed - the seeded culture has grown. The broth in the test tube, where the bacteriophage was added, remained transparent due to the lysis of the culture under its influence.

Phage typing of bacteria

According to the spectrum of action, the following bacteriophages are distinguished: polyvalent, lysing related types of bacteria; monovalent, lysing bacteria of a certain type; typical, lysing individual types (variants) of bacteria.

For example, one strain of pathogenic staphylococcus can be lysed by several types of phages; therefore, all typical phages (24) and strains of pathogenic staphylococci are combined into 4 groups.

The phage typing method has great importance for epidemiological research, as it allows to identify the source and ways of spreading pathogens. For this purpose, the phagovar of a pure culture isolated from the pathological material is determined on dense nutrient media using typical diagnostic phages.

The fagovar of microorganism culture is determined by the type of phage that caused its lysis. The isolation of bacteria of the same fagovar from different subjects indicates the source of infection.

Phage preparations are used for the treatment and prevention of infectious diseases, as well as in diagnostics - to determine phage sensitivity and phage typing in the identification of microorganisms. The action of phages is based on their strict specificity. The therapeutic and prophylactic effect of phages is due to the lytic activity of the phage itself, as well as the immunizing property of the components (antigens) of destroyed microbial cells in phagolysates, especially in the case of repeated use. When obtaining phage preparations, proven production strains of phages and, accordingly, typical cultures of microorganisms are used. A bacterial culture in a liquid nutrient medium, which is in the logarithmic phase of reproduction, is infected with a phage mother suspension.

The phage-lysed culture (usually the next day) is filtered through bacterial filters and a quinosol solution is added as a preservative to the phage-containing filtrate.
The finished phage preparation is clear liquid yellowish color. For longer storage, some phages are available in dry form (in tablets). In the treatment and prevention of intestinal infections, phages are used simultaneously with a solution of sodium bicarbonate, since the acidic contents of the stomach destroy the phage. The phage does not remain in the body for long (5-7 days), so it is recommended to reapply.

In the Soviet Union, the following drugs were produced for the treatment and prevention of diseases: typhoid, salmo-pella, dysentery, coliphage, staphylococcal phage and streptococcal. Currently, phages are used for treatment and prevention in combination with antibiotics. This application has a more effective effect on antibiotic-resistant forms of bacteria.

Diagnostic bacteriophages are widely used to identify bacteria isolated from a patient or from infected environmental objects. With the help of bacteriophages, due to their high specificity, it is possible to determine the types of bacteria and, with greater accuracy, individual types of isolated bacteria. Phage diagnostics and phage typing of bacteria of the genus Salmonella, Vibrio and staphylococci have been developed. Phage typing helps to establish the source of infection, study epidemiological relationships, and distinguish between sporadic and epidemic cases of diseases.
Phage diagnostics and phage typing are based on the principle of joint cultivation of an isolated microorganism with the corresponding species or type phages. positive result the presence of a well-pronounced lysis of the studied culture with the species phage, and then with one of the typical phages is considered.

CM. ZAKHARENKO, Candidate of Medical Sciences, Associate Professor, Military-medical Academy them. CM. Kirov, St. Petersburg

Bacteriophages are unique microorganisms, on the basis of which a special group of therapeutic and prophylactic preparations has been created in terms of their properties and characteristics. The natural physiological mechanisms of interaction between phages and bacteria underlying their action make it possible to predict the infinite diversity of both bacteriophages themselves and possible ways their applications. As bacteriophage collections expand, new target pathogens will undoubtedly appear, and the range of diseases in which phages can be used both as monotherapy and as part of complex treatment regimens will expand.

Yes, use polyvalent pyobacteriophage Sextaphage in the treatment of infected pancreatic necrosis (Perm State Medical Academy named after Academician E.A. Wagner) made it possible to quickly restore the main parameters of homeostasis and the functions of organs and systems in patients. The number of postoperative complications and deaths also significantly decreased: in the group of patients who received standard therapy, mortality was 100%, while in the troupe who received BF, it was 16.6%.

Due to the harmlessness and reactogenicity of BF preparations, it is possible to use them in pediatric practice, including in newborns. The experience of the Nizhny Novgorod Children's Regional Clinical Hospital is interesting, where during the period of complication of the epidemiological situation, along with the usual anti-epidemic measures, BP - Intesti-bacteriophage and BP Pseucfomonas aeruginosa were used. An 11-fold decrease in the incidence of nosocomial infection of Pseudomonas aeruginosa showed the high efficiency of BP use. BF preparations can be prescribed both for the treatment of dysbacteriosis and disorders digestive system and to prevent mucosal colonization gastrointestinal tract opportunistic bacteria. Multicomponent preparations of BF are ideal for immediate relief of the first signs of gastrointestinal upset.

To date, the company has planned a number of priority areas development and production of therapeutic and prophylactic bacteriophages, which correlate with the newly emerging global trends. New preparations are being created and introduced: BF against serrations and enterobacteria have been developed, work is underway to create a phage preparation against Helicobacter pylori.

Only one manufacturer of these drugs - NPO Microgen, according to the report of the deputy head of the department of science and innovative development Alla Lobastova, produces more than 2 million packages annually. Unfortunately, the ideas of many doctors about bacteriophages are far from being objective. Not many people know that bacteriophages active against the same pathogen can belong to different families, have different life cycles, etc. For example, P. aeruginosa bacteriophages belong to the families Myoviridae, Podoviridae, Siphoviridae, life cycle or moderate. Different strains of the same pathogen may have different susceptibility to bacteriophages. Most experts know (heard, someone used) about the existence of liquid and tablet dosage forms of therapeutic and prophylactic preparations of bacteriophages. However, their spectrum is much broader, which can be attributed to unconditional advantages, especially in combination with a variety of routes of administration (ingestion, administration in enemas, applications, irrigation of wounds and mucous membranes, introduction into wound cavities, etc.). The obvious advantages of bacteriophages traditionally include a specific effect on a rather limited population of bacteria, a limited time existence (until the target population of microorganisms disappears), the absence of such side effects as toxic and allergic reactions, dysbiotic reactions, etc. These drugs can be used in a variety of age groups and during pregnancy. Bacteriophages themselves are not significant allergens. Cases of intolerance to bacteriophage preparations are mostly associated with a reaction to the components of the nutrient medium. All major manufacturers of this group of drugs strive for the maximum quality of the components used, which reduces the likelihood of such reactions. In the context of growing antibiotic resistance, some authors propose to consider bacteriophages as the best alternative antibiotics. Therapeutic and prophylactic preparations of bacteriophages are a cocktail of specially selected combinations (a complex of polyclonal highly virulent bacterial viruses specially selected against the most common groups of pathogens of bacterial infections) based on the manufacturer's phage collections. Branches of Federal State Unitary Enterprise NPO Microgen in Ufa, Perm and Nizhny Novgorod are modern centers for the production of such drugs. Ability to create customized pathogenic microorganisms therapeutic and prophylactic preparations of bacteriophages is another major advantage of this group of preparations. The growth of bacterial resistance to antimicrobial drugs and the often occurring polyetiology of modern infectious diseases require combined antibiotic therapy (two, three, and sometimes more antimicrobials). For selection efficient scheme therapy with antibiotics, in addition to the actual sensitivity of the bacteria to the drug, it is necessary to take into account enough big number factors. Phage therapy also has certain advantages in this respect. On the one hand, the use of a combination of bacteriophages is not accompanied by their interaction with each other and does not lead to a change in the schemes of their application. Within the existing set of therapeutic bacteriophages, there are a number of well-proven combinations - bacteriophage coliproteus, pyobacteriophage polyvalent, intesti-bacteriophage. On the other hand, bacteria do not common mechanisms resistance to antibiotics and phages, therefore, they can be used both when the pathogen is resistant to one of the drugs, and in the combination of "antibiotic + bacteriophage". This combination is especially effective for destroying microbial biofilms. The experiment convincingly shows that combined application iron antagonists and bacteriophage can disrupt the formation of Klebsiella pneumoniae biofilms. At the same time, both a significant decrease in the number of microbial populations and a decrease in the number of "young" cells are noted. One more important feature action of bacteriophages is such a phenomenon as the induction of apoptosis. Some strains of E. coli have genes that cause cell death after the introduction of the T4 bacteriophage into it. Thus, in response to the expression of the late genes of the T4 phage, the lit gene (encodes a protease that destroys the EF-Tu elongation factor necessary for protein synthesis) blocks the synthesis of all cellular proteins. The prrC gene encodes a nuclease that cleaves lysine tRNA. The nuclease is activated by the product of the T4 phage stp gene. In T4 phage-infected cells, rex genes (belonging to the phage genome and expressed in lysogenic cells) cause the formation of ion channels, leading to the loss of vital ions by the cells and subsequently to death. The T4 phage itself can prevent cell death by closing the channels with its proteins, products of the rII genes. In the case of the formation of bacterial resistance to an antibiotic, one has to look for new options for modifying the active molecule or fundamentally new substances. Unfortunately for last years the pace of introduction of new antibiotics has slowed significantly. The situation with bacteriophages is fundamentally different. The collections of major manufacturers include dozens of ready-made bacteriophage strains and are constantly replenished with new active phages. Thanks to constant monitoring of the sensitivity of isolated pathogens to bacteriophages, manufacturers adjust the phage compositions supplied to the regions. Thanks to adapted bacteriophages, it is possible to eliminate outbreaks of nosocomial infections caused by antibiotic-resistant strains.

At oral intake bacteriophages quickly reach the foci of infection localization: when taken orally by patients with purulent-inflammatory diseases, after an hour, phages enter the bloodstream, after 1–1.5 hours they are detected from bronchopulmonary exudate and from the surface of burn wounds, after 2 hours - from urine, and also from the cerebrospinal fluid of patients with traumatic brain injury.

Thus, bacteriophages are unique microorganisms, on the basis of which a special group of therapeutic and prophylactic preparations has been created in terms of their properties and characteristics. The natural physiological mechanisms of interaction between phages and bacteria underlying their action make it possible to predict an infinite variety of both bacteriophages themselves and possible ways of using them. As bacteriophage collections expand, new target pathogens will undoubtedly appear, and the range of diseases in which phages can be used both as monotherapy and as part of complex treatment regimens will expand. A modern take on further fate phage therapy should be based both on the high specificity of their action and on the need to strictly observe all the rules of phage therapy. Contrasting bacteriophages with any means of etiotropic therapy is erroneous.

In the middle of the last century, biological science took a revolutionary step forward by establishing the molecular basis for the functioning of living systems. A huge role in the successful research that led to the determination of the chemical nature of hereditary molecules, the decoding of the genetic code and the creation of gene manipulation technologies was played by bacteriophages discovered at the beginning of the last century. To date, these bacterial viruses have mastered many useful “professions” for humans: they are used not only as safe antibacterial drugs, but also as disinfectants, and even as the basis for creating electronic nanodevices.

When in the 1930s a group of scientists took up the problems of the functioning of living systems, then in search of the simplest models they paid attention to bacteriophages- bacterial viruses. After all, among biological objects there is nothing simpler than bacteriophages, besides, they can be easily and quickly grown and analyzed, and viral genetic programs are small.

A phage is a minimally sized natural structure containing a densely packed genetic program (DNA or RNA), in which there is nothing superfluous. This program is enclosed in a protein shell, equipped with a minimum set of devices for its delivery inside the bacterial cell. Bacteriophages cannot reproduce by themselves, and in this sense they cannot be considered full-fledged living objects. Their genes begin to work only in bacteria, using the biosynthetic systems available in the bacterial cell and the reserves of molecules necessary for synthesis. However, the genetic programs of these viruses do not fundamentally differ from the programs of more complex organisms, so experiments with bacteriophages made it possible to establish fundamental principles the structure and operation of the genome.

Subsequently, this knowledge and the methods developed during the research became the foundation for the development of biological and medical science, as well as a wide range of biotechnological applications.

Fighters against pathogens

The first attempts to use bacteriophages for the treatment of infectious diseases were made almost immediately after their discovery, but the lack of knowledge and imperfect biotechnologies of that time did not allow them to achieve full success. Nevertheless, further clinical practice showed the fundamental possibility of the successful use of bacteriophages in infectious diseases gastrointestinal tract, genitourinary system, in acute purulent-septic conditions of patients, for the treatment of surgical infections, etc.

Compared to antibiotics, bacteriophages have a number of advantages: they do not cause side effects, moreover, they are strictly specific for certain types of bacteria, so when they are used, the normal human microbiome is not disturbed. However, such high selectivity also creates problems: in order to successfully treat a patient, it is necessary to know exactly the infectious agent and select the bacteriophage individually.

Phages can also be used prophylactically. For example, the Moscow Research Institute of Epidemiology and Microbiology. G. N. Gabrichevsky developed the prophylactic product "FOODFAG" based on a cocktail of bacteriophages, which reduces the risk of infection with acute intestinal infections. Clinical studies have shown that a weekly intake of the drug allows you to get rid of hemolyzing Escherichia coli and other pathogenic and opportunistic bacteria causing intestinal dysbiosis.

Bacteriophages treat infectious diseases not only of people, but also of domestic and farm animals: mastitis in cows, colibacillosis and escherichiosis in calves and pigs, salmonellosis in chickens ... It is especially convenient to use phage preparations in the case of aquaculture - for the treatment of industrially grown fish and shrimp, because they stay in water for a long time. Bacteriophages also help to protect plants, although the use of phage technologies in this case is difficult due to the impact natural factors, such as sunlight and rain, are detrimental to viruses.

Phages can play a big role in maintaining the microbiological safety of food, since the use of antibiotics and chemical agents in the food industry does not solve this problem, while reducing the level of environmental friendliness of products. The seriousness of the problem itself is evidenced by statistics: for example, in the United States and Russia, up to 40 thousand cases of salmonellosis are registered annually, of which 1% die. The spread of this infection is largely associated with the rearing, processing and consumption of various types of poultry, and attempts to use bacteriophages to combat it have shown promising results.

Yes, an American company Intralytix manufactures phage preparations to combat listeriosis, salmonellosis and bacterial contamination by Escherichia coli. They are approved for use as additives to prevent the growth of bacteria on food - they are sprayed on meat and poultry products, as well as vegetables and fruits. Experiments have shown that a cocktail of bacteriophages can also be successfully used in the transportation and sale of live pond fish to reduce bacterial contamination not only of water, but also of the fish itself.

An obvious application of bacteriophages is disinfection, that is, the destruction of bacteria in places where they should not be: in hospitals, on food production etc. For this purpose, a British company Fixed Phage developed a method for fixing phage preparations on surfaces, which ensures the preservation of biological activity phages up to three years.

Bacteriophages - "Drosophila" of molecular biology

In 1946, at the 11th symposium in the famous American laboratory at Cold Spring Harbor, the theory of "one gene - one enzyme" was proclaimed. Bacteriologist A. Hershey and "former" physicist, molecular biologist M. Delbrück reported on the exchange of genetic traits between different phages while simultaneously infecting Escherichia coli cells. This discovery, made at a time when the physical carrier of the gene was not yet known, testified that the phenomenon of "recombination" - the mixing of genetic traits, is characteristic not only of higher organisms, but also of viruses. The discovery of this phenomenon subsequently made it possible to study in detail the molecular mechanisms of replication. Later, experiments with bacteriophages made it possible to establish the principles of the structure and operation of genetic programs.

In 1952, A. Hershey and M. Chase experimentally proved that the hereditary information of bacteriophage T2 is encoded not in proteins, as many scientists believed, but in DNA molecules (Hershey & Chase, 1952). The researchers followed the reproduction process in two groups of bacteriophages, one carrying radiolabeled proteins and the other carrying DNA molecules. After infection of bacteria with such phages, it turned out that only viral DNA is transmitted to the infected cell, which served as evidence of its role in the storage and transmission of hereditary information.

In the same year, American geneticists D. Lederberg and N. Zindler, in an experiment involving two strains of Salmonella and the P22 bacteriophage, found that the bacteriophage is capable of incorporating DNA fragments of the host bacterium during reproduction and transmitting them to other bacteria upon infection (Zinder & Lederberg , 1952). This phenomenon of gene transfer from a donor bacterium to a recipient bacterium has been termed "transduction". The results of the experiment became another confirmation of the role of DNA in the transmission of hereditary information.

In 1969, A. Hershey, M. Delbrück and their colleague S. Luria became Nobel laureates "for their discoveries concerning the mechanism of replication and the genetic structure of viruses."

In 1972, while studying the process of replication (copying cellular information) of E. coli DNA, R. Bird and colleagues used bacteriophages as probes capable of integrating into the bacterial cell genome and found that the replication process proceeds in two directions along the chromosome (Stent, 1974 ).

Seven Days of Creation

Modern methods of synthetic biology make it possible not only to make various modifications to phage genomes, but also to create completely artificial active phages. Technologically, this is not difficult, you just need to synthesize the phage genome and introduce it into a bacterial cell, and there it will start all the processes necessary for the synthesis of proteins and the assembly of new phage particles. In modern laboratories, this work will take only a few days.

Genetic modifications are used to change the specificity of phages and increase their efficiency. therapeutic action. To do this, the most aggressive phages are provided with recognition structures that bind them to the target bacteria. Also, genes encoding toxic proteins for bacteria that disrupt metabolism are additionally inserted into viral genomes - such phages are more deadly for bacteria.

Bacteria have several defense mechanisms against antibiotics and bacteriophages, one of which is the destruction of viral genomes. restriction enzymes acting on specific nucleotide sequences. To increase the therapeutic activity of phages, due to the degeneracy of the genetic code, the sequences of their genes can be “reformatted” in such a way as to minimize the number of nucleotide sequences that are “sensitive” to enzymes, while maintaining their coding properties.

A universal way to protect bacteria from all external influences - the so-called biofilms, films of DNA, polysaccharides, and proteins that bacteria create together and where neither antibiotics nor therapeutic proteins penetrate. Such biofilms are headache doctors, as they contribute to the destruction of tooth enamel, are formed on the surface of implants, catheters, artificial joints, as well as in respiratory tract, on the surface of the skin, etc. To combat biofilms, special bacteriophages were constructed containing a gene encoding a special lytic enzyme that destroys bacterial polymers.

Enzymes "from bacteriophage"

A large number of enzymes that are widely used today in molecular biology and genetic engineering were discovered as a result of research on bacteriophages.

One such example is the restriction enzymes, a group of bacterial nucleases that cleave DNA. Back in the early 1950s. It was found that bacteriophages isolated from cells of one strain of bacteria often reproduce poorly in a closely related strain. The discovery of this phenomenon meant that bacteria have a system for suppressing the reproduction of viruses (Luria & Human, 1952). As a result, an enzymatic restriction-modification system was discovered, with the help of which bacteria destroyed foreign DNA that had entered the cell. The isolation of restriction enzymes (restriction endonucleases) gave molecular biologists an invaluable tool to manipulate DNA: insert one sequence into another or cut out the necessary chain fragments, which ultimately led to the development of recombinant DNA technology.

Another enzyme widely used in molecular biology is bacteriophage T4 DNA ligase, which “crosslinks” the “sticky” and “blunt” ends of double-stranded DNA and RNA molecules. And recently, genetically modified variants of this enzyme with greater activity have appeared.

Most of the RNA ligases used in laboratory practice, which "sew" single-stranded RNA and DNA molecules, also originate from bacteriophages. In nature, they mainly serve to repair broken RNA molecules. Researchers most commonly use bacteriophage T4 RNA ligase, which can “sew” single-stranded polynucleotides onto RNA molecules to label them. This technique is used to analyze the structure of RNA, search for RNA-protein binding sites, oligonucleotide synthesis, etc. Recently, thermostable RNA ligases isolated from bacteriophages rm378 and TS2126 have appeared among routinely used enzymes (Nordberg Karlsson, et al., 2010; Hjorleifsdottir , 2014).

From bacteriophages, some of another group of extremely important enzymes, polymerases, were also obtained. For example, the very "precise" bacteriophage T7 DNA polymerase, which has found application in various areas of molecular biology, such as site-directed mutagenesis, but is mainly used to determine the primary structure of DNA.

A chemically modified T7 phage DNA polymerase has been proposed as perfect tool for DNA sequencing as early as 1987 (Tabor & Richardson, 1987). The modification of this polymerase has increased its efficiency by several times: the rate of DNA polymerization in this case reaches more than 300 nucleotides per second, so it can be used to amplify large DNA fragments. This enzyme became the precursor of sequenase, a genetically engineered enzyme optimized for DNA sequencing in the Sanger reaction. Sequenase is different high efficiency and the ability to incorporate nucleotide analogs into the DNA sequence that are used to improve sequencing results.

The origin of bacteriophages is also the main RNA polymerases used in molecular biology (DNA-dependent RNA polymerases) - enzymes that catalyze the transcription process (reading RNA copies from the DNA template). These include SP6, T7, and T3 RNA polymerases, named after the respective bacteriophages SP6, T7, and T3. All these enzymes are used for in vitro synthesis of antisense RNA transcripts, labeled RNA probes, etc.

The first fully sequenced DNA genome was the φ174 phage genome, over 5000 nucleotides long (Sanger et al., 1977). This decoding was carried out by a group of English biochemist F. Sanger, the creator of the famous DNA sequencing method of the same name.

Polynucleotide kinases catalyze the transfer of a phosphate group from an ATP molecule to the 5' end of a nucleic acid molecule, the exchange of 5' phosphate groups, or the phosphorylation of the 3' ends of mononucleotides. In laboratory practice, bacteriophage T4 polynucleotide kinase is most widely used. It is commonly used in experiments to label DNA with a radioactive isotope of phosphorus. Polynucleotide kinase is also used to search for restriction sites, DNA and RNA fingerprinting, the synthesis of substrates for DNA or RNA ligases.

In molecular biological experiments, bacteriophage enzymes such as T4 phage polynucleotide kinase, which is commonly used for labeling DNA with a radioactive isotope of phosphorus, DNA and RNA fingerprinting, etc., as well as enzymes that cleave DNA, which are used to obtain single-stranded DNA templates, are also widely used in molecular biological experiments. for sequencing and analysis of nucleotide polymorphism.

Using the methods of synthetic biology, it was also possible to develop bacteriophages armed with the most sophisticated weapons that bacteria use against the phages themselves. It's about about bacterial CRISPR-Cas systems, which are a complex of the nuclease enzyme that cleaves DNA and the RNA sequence that directs the action of this enzyme to a specific fragment of the viral genome. A piece of phage DNA serves as a “pointer”, which the bacterium stores “for memory” in a special gene. When a similar fragment is found inside a bacterium, this protein-nucleotide complex destroys it.

Having figured out the mechanism of operation of CRISPR-Cas systems, the researchers tried to equip the phages themselves with a similar “weapon”, for which a complex of genes encoding a nuclease and addressing RNA sequences complementary to specific regions of the bacterial genome was introduced into their genome. The "target" can be the genes responsible for multiple drug resistance. The experiments were crowned with complete success - such phages with great efficiency affected the bacteria to which they were "tuned".

Phage antibiotics

For therapeutic purposes, phages do not have to be used directly. Over millions of years of evolution, bacteriophages have developed an arsenal of specific proteins - tools for recognizing target microorganisms and manipulating the biopolymers of the victim, on the basis of which antibacterial drugs can be created. The most promising proteins of this type are the endolysin enzymes, which phages use to destroy the cell wall upon exiting the bacterium. By themselves, these substances are powerful. antibacterial agents, non-toxic to humans. The efficiency and direction of their action can be increased by changing the addressing structures in them - proteins that specifically bind to certain bacteria.

Most bacteria are divided according to the structure of the cell wall into gram-positive, the membrane of which is covered with a very thick layer of peptidoglycans, and gram-negative, in which the peptidoglycan layer is located between two membranes. The use of natural endolysins is especially effective in the case of gram-positive bacteria (staphylococci, streptococci, etc.), since their peptidoglycan layer is located outside. Gram-negative bacteria (Pseudomonas aeruginosa, Salmonella, Escherichia coli, etc.) are a less accessible target, since the enzyme must penetrate the outer bacterial membrane to reach the inner peptidoglycan layer.

To overcome this problem, the so-called artilysins were created - modified variants of natural endolysins containing polycationic or amphipathic peptides that destabilize the outer membrane and ensure the delivery of endolysin directly to the peptidoglycan layer. Artilysins have a high bactericidal activity and have already been shown to be effective in the treatment of otitis media in dogs (Briers et al., 2014).

An example of a modified endolysin that selectively acts on certain bacteria is the drug P128 of the Canadian company Ganga Gen Inc.. It is a biologically active fragment of endolysin connected to lysostaphin, a targeting protein molecule that binds to the surface of staphylococcal cells. The resulting chimeric protein has high activity against various strains of staphylococcus, including those with multidrug resistance.

"Counters" of bacteria

Bacteriophages serve not only as a versatile therapeutic and "disinfectant" agent, but also as a convenient and accurate analytical tool for a microbiologist. For example, due to their high specificity, they are natural analytical reagents for the detection of bacteria of a certain type and strain.

In the simplest version of such a study, various diagnostic bacteriophages are added dropwise to a Petri dish with a nutrient medium inoculated with a bacterial culture. If the bacterium turns out to be sensitive to the phage, then at this place of the bacterial "lawn" a "plaque" is formed - a transparent area with killed and lysed bacterial cells.

By analyzing the multiplication of phages in the presence of target bacteria, one can quantify the abundance of the latter. Since the number of phage particles in the solution will increase in proportion to the number of bacterial cells contained in it, it is sufficient to determine the titer of the bacteriophage to estimate the number of bacteria.

The specificity and sensitivity of such an analytical reaction is quite high, and the procedures themselves are simple to perform and do not require sophisticated equipment. It is important that diagnostic systems based on bacteriophages signal the presence of a living pathogen, while other methods, such as PCR and immunoanalytical methods, only indicate the presence of biopolymers belonging to this bacterium. This type of diagnostic methods are particularly suitable for use in environmental studies, as well as in the food industry and agriculture.

Now, special methods are used to identify and quantify different strains of microorganisms. reference species phages. Very fast, working almost in real time analytical systems can be created on the basis of genetically modified bacteriophages, which, when they enter a bacterial cell, trigger the synthesis of reporter fluorescent (or capable of luminescence) proteins, such as luciferase. When the necessary substrates are added to such a medium, a luminescent signal will appear in it, the value of which corresponds to the content of bacteria in the sample. Such "light-labeled" phages have been developed to detect dangerous pathogens - the causative agents of plague, anthrax, tuberculosis, and plant infections.

Probably, with the help of modified phages, it will also be possible to solve the long-standing problem of global importance - to develop cheap and quick methods detection of causative agents of tuberculosis early stage diseases. This task is very difficult, since mycobacteria, causing tuberculosis, are characterized by extremely slow growth when cultivated in laboratory conditions. Therefore, the diagnosis of the disease traditional methods may take up to several weeks.

Phage technology makes this task easier. Its essence is that bacteriophage D29 is added to the samples of the analyzed blood, capable of infecting wide range mycobacteria. The bacteriophages are then separated, and the sample is mixed with a rapidly growing non-pathogenic culture of mycobacteria, also sensitive to this bacteriophage. If initially there were mycobacteria in the blood that were infected with phages, then the production of bacteriophage will also be observed in the new culture. In this way, single cells of mycobacteria can be detected, and the diagnostic process itself is reduced from 2–3 weeks to 2–5 days (Swift & Rees, 2016).

Phage display

Today, bacteriophages are widely used as simple systems for the production of proteins with desired properties. This is the one developed in the 1980s. extremely effective molecular selection technique - phage display. This term was proposed by the American J. Smith, who proved that on the basis of Escherichia coli bacteriophages, it is possible to create a viable modified virus that carries a foreign protein on its surface. To do this, the corresponding gene is introduced into the phage genome, which merges with the gene encoding one of the surface viral proteins. Such modified bacteriophages can be isolated from a mixture with wild-type phages due to the ability of a "foreign" protein to bind to specific antibodies (Smith, 1985).

From Smith's experiments followed two important findings: firstly, using recombinant DNA technology, it is possible to create populations of 10 6 -10 14 phage particles, huge in diversity, each of which carries on its surface different variants proteins. Such populations are called combinatorial phage libraries. Secondly, by isolating a specific phage from a population (for example, having the ability to bind to a certain protein or organic molecule), this phage can be propagated in bacterial cells and get an unlimited number of children with the given properties.

Phage display today produces proteins that can selectively bind to therapeutic targets, such as those exposed on the surface of the M13 phage that can recognize and interact with tumor cells. The role of these proteins in the phage particle is to “package” the nucleic acid, so they are well suited for creating gene therapy drugs, only in this case they form a particle already with a therapeutic nucleic acid.

Today, there are two main areas of application of phage display. Peptide-based technology is being used to explore receptors and map antibody binding sites, design immunogens and nanovaccines, and map substrate binding sites for enzyme proteins. Technology based on proteins and protein domains - for the selection of antibodies with desired properties, the study of protein-ligand interactions, screening of expressed complementary DNA fragments and targeted modifications of proteins.

Using phage display, it is possible to introduce recognition groups into all types of surface viral proteins, as well as into the main protein that forms the bacteriophage body. By introducing peptides with desired properties into surface proteins, a whole range of valuable biotechnological products can be obtained. For example, if this peptide mimics a protein dangerous virus or bacteria, recognizable immune system, then such a modified bacteriophage is a vaccine that can be easily, quickly and safely developed.

If the terminal surface protein of the bacteriophage is “addressed” to cancer cells, and reporter groups (for example, fluorescent or magnetic) are attached to another surface protein, then a means for detecting tumors will be obtained. And if a cytotoxic drug is also attached to the particle (and modern bioorganic chemistry makes it easy to do this), then we get a drug that targets cancer cells.

One of the important applications of protein phage display is the creation of phage libraries of recombinant antibodies, where antigen-binding fragments of immunoglobulins are located on the surface of fd or M13 phage particles. Human antibody libraries are of particular interest because such antibodies can be used in therapy without limitation. In recent years, about a dozen therapeutic antibodies constructed using this method have been sold on the US pharmaceutical market alone.

"Industrial" phages

The phage display methodology has also found quite unexpected applications. After all, bacteriophages are primarily nanosized particles of a certain structure, on the surface of which proteins are located, which, using a phage display, can be “provided” with the properties to specifically bind to the desired molecules. Such nanoparticles open the widest possibilities to create materials with a given architecture and "smart" molecular nanodevices, while their production technologies will be environmentally friendly.

Since the virus is a fairly rigid structure with a certain ratio of dimensions, this circumstance makes it possible to use it to obtain porous nanostructures with a known surface area and a desired distribution of pores in the structure. As is known, the surface area of ​​a catalyst is the critical parameter determining its efficiency. And the existing technologies for the formation of the thinnest layer of metals and their oxides on the surface of bacteriophages make it possible to obtain catalysts with an extremely developed regular surface of a given dimension. (Lee et al., 2012).

MIT researcher A. Belcher used bacteriophage M13 as a template for the growth of rhodium and nickel nanoparticles and nanowires on the surface of cerium oxide. The resulting catalyst nanoparticles facilitate the conversion of ethanol to hydrogen; thus, this catalyst can be very useful for upgrading existing and creating new hydrogen fuel cells. A catalyst grown on a virus template differs from a “conventional” catalyst of similar composition in higher stability, it is less prone to aging and surface deactivation (Nam et al. . , 2012).

By coating filamentous phages with gold and indium dioxide, electrochromic materials were obtained - porous nanofilms that change color when the electric field changes, capable of responding to a change in the electric field one and a half times faster than known analogues. Such materials are promising for creating energy-saving ultra-thin screen devices (Nam et al., 2012).

In Massachusetts Institute of Technology bacteriophages became the basis for the production of very powerful and extremely compact electric batteries. For this, live, genetically modified M13 phages were used, which are harmless to humans and capable of attaching various metal ions to the surface. As a result of the self-assembly of these viruses, structures of a given configuration were obtained, which, when coated with a metal, formed rather long nanowires, which became the basis of the anode and cathode. When self-forming the anode material, a virus capable of attaching gold and cobalt oxide was used, for the cathode - capable of attaching iron phosphate and silver. The last phage also possessed the ability to "pick up" the ends of a carbon nanotube due to molecular recognition, which is necessary to ensure efficient electron transfer.

Materials for solar cells have also been created based on complexes of the bacteriophage M13, titanium dioxide, and single-walled carbon nanotubes (Dang et al., 2011).

Recent years have been marked by extensive research on bacteriophages, which are finding new applications not only in therapy, but also in bio- and nanotechnologies. Their obvious practical result should be the emergence of a new powerful direction of personalized medicine, as well as the creation of a whole range of technologies in the food industry, veterinary medicine, agriculture and in the production of modern materials. We expect that the second century of bacteriophage research will bring as many discoveries as the first.

Literature
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