RU2730616C1 - Antibacterial composition (embodiments) and use of protein as an antimicrobial agent directed against acinetobacter baumannii bacteria, (embodiments) - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, specifically to therapeutic proteins, and can be used in medicine as an antibacterial agent. Disclosed is use of recombinant endolysin bacteriophage, including in combination with pharmaceutically acceptable carriers and/or substances, increasing permeability of membranes, as an antimicrobial agent directed against bacteria Acinetobacter baumannii. Antibacterial compositions are obtained based on said protein, including fused with 8-histidine tag.EFFECT: invention provides effective penetration of endolysin without adding various permeabilisers or covalently bound penetrating peptides through the outer membrane of said bacteria and cleavage of peptidoglycan, which ultimately leads to death of bacteria.4 cl, 3 tbl, 6 ex, 5 dwg

Description

The technical field to which the invention relates

The invention relates to biochemistry, molecular biology, microbiology and biotechnology and can be used in the creation of biological preparations for the prevention and treatment of infections, as well as disinfection.

State of the art

Bacterial resistance to existing antimicrobial agents is one of the major health problems of our time. Horizontal gene transfer allows bacteria to very quickly spread new mechanisms of resistance, which, along with the misuse and abuse of antibiotics in the population, as well as in agriculture and industry, leads to the emergence of strains that are generally resistant to antibiotics. The World Health Organization pays special attention to pathogens of the ESKAPE group, which include Enterococcus faecium , Staphylococcus aureus , Acinetobacter baumannii , Pseudomonas aeruginosa , Klebsiella pneumoniae and other representatives of Enterobacteriaceae . These bacteria are able to acquire resistance to several antibacterial agents at once, and more often than others cause infections that are life-threatening to patients, including nosocomial ones.

An alternative to antibiotics and synthetic antibacterial agents are bacteriophages, the use of which began even before the discovery of penicillin. The use of bacteriophages has many advantages: automatic dosing depending on the degree of infection, minimal disruption of natural microflora, low probability of resistance development, compatibility with antibiotics, rapid development of phage-based drugs and their low cost, destruction of biofilms, the possibility of one-time administration and the use of low doses as well as a low degree of pressure on the environment.

In addition to the bacteriophage preparations of the Microgen company registered in the Russian Federation, there are many isolated bacteriophages that can be used to treat infections caused by gram-negative bacteria. For example, the virulent phage Phi 03 (RU 2112800, published 03/29/1996) has the ability to act on Pseudomonas aeruginosa cells that have pili. However, its spectrum of action is rather narrow. Phage Phi 02 (RU 2113476, published 03/29/1996) has a wide spectrum of action. Bacteriophage ph57 (RU 2455355, published on April 29, 2011) also has a wide spectrum and can be used to treat purulent-septic infections. The virulent bacteriophage strain Acinetobacter baumannii AP22 (RU 2439151, published on June 24, 2010) has a narrow spectrum of action and is capable of lyzing only representatives of the species Acinetobacter baumannii . Bacteriophage Esc-COP-1 is able to specifically destroy strains of Shiga-toxin producing E. coli type F18 and can be used in the medical industry (RU 2662985, published on December 28, 2015). The highly virulent Pseudomonas aeruginosa ph57 bacteriophage strain has a wide spectrum of lytic activity against Pseudomonas aeruginosa bacteria and can be used as a basis for the preparation of an antiseptic agent against Pseudomonas aeruginosa (RU 2455355, published on 19.04.2011). The bacteriophage strain Salmonella typhimurium S-394 has polyvalent lytic activity against bacteria of the genera Escherichia coli , the Salmonella TA 1537 and Salmonella TA 100 strains and the Shigella sonnei 32 and Shigella flexneri Ad strains (RU 2412243, published on June 16, 2009). It is also possible to note antibacterial compositions of wide action sperm containing several bacteriophages, for example, those described in patents RU 2518303 (published on June 29, 2012) and RU 2628312 (published on March 16, 2015).

However, there are also disadvantages of using drugs based on bacteriophages. It is difficult and takes a long time to isolate bacteriophages from bacterial cultures, they can transfer genes of bacterial toxins, bacteria can often become resistant to them, they have strong immunogenicity. Also, their isolation can be difficult, and it is difficult, and sometimes impossible, to normalize their pharmacokinetic characteristics. For wound infections, it was shown that the presence of bacteriophages caused an unproductive immune response, contributing to the development of chronic wound infection and determining a more severe course.

It is believed that a more promising class of antibacterial agents can be bacteriophage endolysins - lytic enzymes capable of cleaving peptidoglycan of the bacterial cell wall, which entails the death of bacteria due to hypotonic lysis.

Endolysins have several advantages over bacteriophages and antibacterial molecules. Most often, they act specifically on a certain type of bacteria, while the lytic effect is achieved very quickly due to the direct action on peptidoglycan. However, for many endolysins, whose action has been shown against gram-negative bacteria, a rather wide spectrum of activity is found against various genera of representatives of gram-negative pathogens. Other advantages of endolysins include a low probability of developing resistance, an effect on antibiotic-resistant strains, as well as on bacteria in any metabolic stage, including dormant ones.

Endolysins are increasingly being noted as potential and effective drugs in the fight against various pathogens. Independent groups of scientists are conducting research on the creation and application of them to combat gram-negative bacteria of the especially important ESKAPE group. At the same time, very often (but not always) the use of pure endolysins is hindered by the presence of an outer membrane that shields peptidoglycan from the action of the enzyme. Polymyxins, polyguanidines and their derivatives, aminoglycosides, ethylenediamine tetraacetic acid (EDTA) and its salts, citric acid, etc. can be used as substances that increase the permeability of the outer membrane.

Among the examples of endolysins, whose bactericidal effect is manifested or significantly increased in the presence of permeabilizing agents, in particular EDTA, are the antipseudomonal endolysins EL188 (WO 2015071437 A1, published 05.21.2015) and OBPgpLYS (US 8846865 B2, published 09.30.2014).

Another approach that allows not to use additional reagents that increase the permeability of outer membranes is the creation of chimeric molecules. These compounds are covalently crosslinked endolysin and a peptide that allows the molecule to be transported across the outer membrane of gram-negative bacteria. In particular, the antimicrobial peptide SMAP-29 was used to create Art-175, which is a modified KZ144 molecule. Unlike KZ144 Art-175 can pass through the outer membrane of the Pseudomonas aeruginosa and destroy bacteria, including those with multiple resistance (US 20160281074 A1, published 09.29.2016).

It is known that OBPgpLYS has also been modified with SMAP-29. At the same time, it begins to exhibit a significantly greater antimicrobial effect on both Pseudomonas aeruginosa strains and other gram-negative bacteria (US 8846865 B2, published 09/30/2014). Modified and unmodified OBPgpLYS are also capable of eliminating bacteria within biofilms (WO 2011134998 A1, published 03.11.2011).

However, there are many examples of recombinant endolysins that are capable of acting on representatives of gram-negative bacteria without permeabilizers or structural modifications.

Endolysin PlyE146, due to the positively charged region at the C-terminus of the protein, also has the ability to penetrate the outer membrane of gram-negative bacteria and lyse the cells of E. coli , P. aeruginosa, and A. baumannii at a concentration of 400 μg / ml (Larpin et al., 2018). Endolysin of bacteriophage Acinetobacter baumannii LysAB2, due to amphiphilic structures in the molecule, has a bactericidal effect against both gram-negative and gram-positive bacteria, the effect is achieved at a concentration of 500 μg / ml. Its spectrum includes strains of A. baumannii , E. coli, and S. aureus (Lai et al., 2011). Also, without the addition of permeabilizers, endolysin P28 at a concentration of 500 μg / ml exhibits its antimicrobial effect; its spectrum of action covers Bacillus subtilis , Bacillus cereus , Klebsiella mobilis , Shigella flexneri , Xanthomonas campestris and Staphylococcus aureus (Dong et al., 2015). Other examples include LysPA26, capable of killing Pseudomonas aeruginosa , Klebsiella pneumonia , Acinetobacter baumannii and Escherichia coli cells at 500 μg / ml (Guo et al., 2017), PlyAB1 with a narrow spectrum of action against A. baumannii (Huang et al. , 2014) and others.

Thus, there are currently many studies on endolysins, whose effect is directed against broad panels of strains of gram-negative bacteria with drug resistance, including multiple. This confirms the relevance of this line of research.

Technical problem

The technical problem is the need to expand the arsenal of lytic agents against gram-negative bacteria, including bacteria with multidrug resistance.

The essence of the invention

This invention provides the recombinant proteins endolysins LysSi3 and LysSt11 (amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, as well as sequences that are at least 90% identical to them, and the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8, as well as sequences that are at least 80% identical to them), which are 8-histidine-tagged recombinant endolysins of bacteriophages SI3 and ST11 (amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, as well as sequences , which are at least 90% identical to them, and the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, as well as sequences that are at least 80% identical to them). They have a broad spectrum of action, showing antibacterial activity against Pseudomonas aeruginosa , Acinetobacter baumannii , Klebsiella pneumoniae , Escherichia coli , Salmonella typhi and other types of bacteria.

Recombinant endolysins LysSi3 and LysSt11 are capable of penetrating the outer membrane without adding additional membrane permeability increasing components and effectively and quickly destroying cells of gram-negative bacteria of various strains, species and genera. They will allow antimicrobial therapy for various types of infections caused by strains of multidrug-resistant bacteria, as well as getting rid of bacteria that have formed biofilms, into which the access of existing antibacterial agents is difficult.

Pseudomonas aeruginosa , Acinetobacter baumannii and Klebsiella pneumoniae are some of the most dangerous causative agents of nosocomial infections, the treatment of which is often difficult due to the high resistance of these types of bacteria to existing drugs, as well as their ability to form biofilms. Infections caused by these pathogens are clinically very diverse, and their course is very severe, especially in the case of nosocomial origin, when the patient's immunity is greatly reduced. According to various estimates, nosocomial infections affect 5-10% of hospitalized patients. Annually in Russia, the number of cases of nosocomial infections recorded is about 2.5 million.

Escherichia coli is a representative of the normal microflora of the body, but among them there are pathogenic varieties that do not differ morphologically from non-pathogenic ones and cause Escherichiosis, most often accompanied by intoxication, fever and damage to the gastrointestinal tract. Infection most often occurs through contaminated water or food. E. coli can also often develop multiple resistance.

Various representatives of the genus Salmonella are pathogens for humans and cause salmonellosis, and in recent years the incidence of salmonellosis has increased dramatically, even in developed countries, as these bacteria are actively developing resistance to the antibiotics used. They can also cause severe nosocomial illnesses.

The complexity of therapy and high mortality indicate the high relevance of the introduction into practice of new effective methods of treating infections caused by gram-negative pathogens.

Endolysins LysSi3 and LysSt11 in pure form, as well as in combinations with various agents that increase membrane permeability, antimicrobial peptides and antimicrobial agents (including antibiotics) can be used in various fields of medicine, veterinary medicine, agriculture and food industry.

These inventions can be used in the form of suitable dosage forms (solution, aerosol, ointment, etc.) for the treatment and prevention of various types of infections caused by gram-negative bacteria sensitive to the action of endolysin. These infections include endocarditis, respiratory infections (pneumonia), central nervous system infections (meningitis and brain abscesses), ear and eye infections (such as otitis media and keratitis), bacteremia, infections of skin structures and soft tissues (including wounds and burns), infections of bones and joints, infections of the urinary tract, infections of the gastrointestinal tract, and others. It is also possible to use drugs based on LysSi3 and LysSt11 for the treatment of pneumonia associated with cystic fibrosis. With the introduction of a preparation containing an effective dose of the active substance, topically, by inhalation, intramuscularly, subcutaneously or intravenously, a favorable outcome of the course of an infectious disease and a rapid elimination of pathogens can be expected.

Also, LysSi3 and LysSt11 can be used for quick and accurate diagnostics in order to determine the causative agent of the infection and the timely initiation of therapy, as well as to determine the sensitivity of various bacterial strains to these enzymes. It is possible to create various detection systems and biosensors based on them.

One of the most important problems in medicine is the formation of biofilms by gram-negative pathogens on various surfaces (including medical instruments). Bacteria inside biofilms are much less susceptible to antimicrobial agents and disinfectants. LysSi3 and LysSt11 in solution or any other suitable form can be used to remove biofilms, as well as to disinfect instruments (especially those used for various surgical procedures), materials, equipment (including ventilators), and surfaces.

In veterinary medicine and agriculture, LysSi3 and LysSt11 can also be used to treat and prevent animal infections caused by susceptible gram-negative bacteria.

In the food industry, LysSi3 and LysSt11 can be used to prevent contamination of water and food with sensitive gram-negative bacteria, as well as to remove biofilms and disinfect various surfaces.

In this case, the technical result is achieved by using antibacterial compositions that contain an effective amount (dose) of the recombinant proteins LysSi3 and LysSt11 and a pharmaceutically acceptable carrier.

The technical result is also achieved by using an antibacterial composition containing an effective amount of endolysins LysSi3 and LysSt11, a permeabilizer and a pharmaceutically acceptable carrier. Polymyxins and their derivatives, aminoglycosides, ethylenediaminetetraacetic acid and its salts (EDTA), citric acid, etc. can be used as permeabilizers.

The term "pharmaceutically acceptable carrier" includes any and all solvents, additives, excipients, adjuvants, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorbent agents, surfactants, propellants, and the like that are pharmaceutically compatible. The carrier (s) must be "acceptable" in terms of harmlessness to the subject to whom the drug is administered based on the subject matter of the claims in the amounts commonly used in drugs. Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unusable for its intended use. In addition, pharmaceutically acceptable carriers are suitable for use without causing serious adverse reactions (such as toxicity, irritation, and allergic reaction). Side effects are "unjustified" when their risk of occurrence outweighs the benefit from the composition. Non-limiting examples of pharmaceutically acceptable carriers or excipients of excipients include any of the standard pharmaceutical carriers such as saline solutions, water, and emulsions such as oil / water emulsions and microemulsions.

The term "effective amount" refers to an amount that, when used or administered at an appropriate frequency of administration or in a specific dosage regimen, is sufficient to prevent or inhibit bacterial growth or prevent the development, improve the course, reduce the severity, duration or progression of an infectious disease, cause regression of the disease or enhances or enhances the prophylactic or therapeutic effect of another treatment, such as antimicrobial therapy.

The term "biofilm" refers to bacteria that attach to surfaces and are immersed in the extracellular matrix they release. A biofilm is a collection of microorganisms where cells adhere to each other on some surface. These adherent cells are often embedded in an extracellular matrix of polymeric material. Biofilm is a polymeric conglomeration usually composed of extracellular DNA, proteins, and polysaccharides.

Illustrations explaining the essence of the invention

The illustrations show explanatory graphic materials that reveal the essence of the invention. These materials do not limit the scope of the applicant's claims and should be used to explain the essence of the claimed technical solution.

FIG. 1 is a graph depicting the bactericidal activity of various concentrations of LysSi3 (Fig. 1A) and LysSt11 (Fig. 1B) against A. baumannii Ts 50-16 strain. As a control, bacterial cells incubated with buffer instead of protein were used. The number of surviving colonies after 18 hours of incubation of Petri dishes is expressed as a decrease in log10 CFU / ml compared to control. For all experiments, mean values of standard deviations are shown after three independent experiments. An asterisk (*) indicates a significant bactericidal effect.

FIG. 2 shows a graph comparing the activity of endolysins LysSi3 (Fig.2A) and LysSt11 (Fig.2B) at concentrations of 10 and 100 μg / ml against A. baumannii Ts 50-16 strain in exponential (Exp) and stationary (St) growth phases without adding and with the addition of 0.5 mM EDTA. The residual enzyme activity (%) is shown in comparison with the control. NS - there is no statistically significant difference from the control culture without the addition of endolysin (p> 0.05, Mann-Whitney test). For all experiments, mean values of standard deviations are shown after three independent experiments. An asterisk (*) indicates a significant bactericidal effect.

FIG. 3 is a graph depicting the effect of pH and 0.5 mM EDTA on antimicrobial activity (FIG. 3A) and LysSt11 (FIG. 3B) at a concentration of 1 μg / ml against A. baumannii Ts 50-16. The residual enzyme activity (%) is shown in comparison with the control. NS - there is no statistically significant difference from the control culture without the addition of endolysin (p> 0.05, Mann-Whitney test). For all experiments, mean values of standard deviations are shown after three independent experiments.

FIG. 4 is a graph of the effect of salts on lytic activity (Fig. 4A) and LysSt11 (Fig. 4B) at a concentration of 10 μg / ml against A. baumannii Ts 50-16. The residual enzyme activity (%) is shown in comparison with the control. NS - there is no statistically significant difference from the control culture without the addition of endolysin (p> 0.05, Mann-Whitney test). All other values are statistically significantly different from the control. For all experiments, mean values of standard deviations are shown after three independent experiments.

FIG. 5 is a graph depicting bactericidal activity (Fig. 5A) and LysSt11 (Fig. 5B) at a concentration of 50 μg / ml against various strains of gram-negative and gram-positive bacteria. The residual enzyme activity (%) is shown in comparison with the control. HA - no bactericidal activity was observed. All values were statistically significantly different from the control culture without endolysin addition (p <0.05, Mann-Whitney test). For all experiments, mean values of standard deviations are shown after three independent experiments.

The following are amino acid and nucleotide sequences: the first of which is the amino acid sequence of bacteriophage SI3 endolysin (SEQ ID NO: 1); the second is the amino acid sequence of bacteriophage ST11 endolysin (SEQ ID NO: 2); the third is the nucleotide sequence of bacteriophage SI3 endolysin (SEQ ID NO: 3); the fourth - the nucleotide sequence of endolysin of bacteriophage ST11 (SEQ ID NO: 4); fifth — amino acid sequence of endolysin LysSi3 modified with 8-histidine tag (SEQ ID NO: 5); the sixth is the amino acid sequence of LysSt11 endolysin modified with an 8-histidine tag (SEQ ID NO: 6); seventh — the nucleotide sequence of LysSi3 endolysin modified with an 8-histidine tag (SEQ ID NO: 7); eighth - the nucleotide sequence of LysSt11 endolysin modified with an 8-histidine tag (SEQ ID NO: 8).

Information confirming the possibility of carrying out the invention

Proteins LysSi3 and LysSt11 (amino acid sequences SEQ ID NO: 5 and SEQ ID NO: 6 and nucleotide sequences SEQ ID NO: 7 and SEQ ID NO: 8) were obtained recombinantly and contain a histidine tag (8 His) in one translation frame. These enzymes are 8-histidine-tagged recombinant bacteriophage endolysins SI3 and ST11 (amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2 and nucleotide sequences SEQ ID NO: 3 and SEQ ID NO: 4) of the Myoviridae family, affecting Salmonella cells typhi . It is predicted that in their structure they contain one domain: a lysozyme-like catalytic (muramidase) domain, which provides bactericidal activity against bacterial cell walls.

Using dynamic laser light scattering, it was revealed that LysSi3 and LysSt11 (proteins with a size of 18.5 kDa) are in solution in the form of particles with a size of about 4 nm, which indicates the stability of protein solutions and being predominantly in the monomeric state.

In terms of antimicrobial properties, LysSi3 and LysSt11 have been shown to have a bactericidal effect on the exponentially growing clinical strain A. baumannii Ts 50-16 at concentrations from 10 μg / ml in 20 mM Tris HCl buffer (pH 7.5 and pH 7.0, respectively). A dose-response plot is shown in FIG. 1.

Under the action of LysSi3 and DnaSt11 on bacteria in the stationary growth phase, the bactericidal effect at a protein concentration of 10 μg / ml and 100 μg / ml disappears, presumably due to changes in the structure of the bacterial cell wall and the absence of effective membrane permeabilization. In this case, the addition of substances that increase membrane permeability (0.5 mM EDTA) promotes the manifestation of the lytic effect, however, it is restored only by 23%, and not to the level of the maximum activity that the enzyme has on bacteria in the exponential growth phase (Fig. 2 ).

In vitro analysis of antibacterial activity at different pH values (from 5.0 to 9.0) showed that in Tris HCl buffer for LysSi3, the most optimal values are weakly acidic (5.0-6.0), as well as neutral and slightly alkaline (7 , 5-8.0). However, at pH 6.5-7.0, a slight decrease in activity is observed, which is presumably associated with a change in the charge of the C-terminus of the protein, which is responsible for effective permeabilization. The addition of 0.5 mM EDTA eliminates the differences in efficacy and eliminates the effect of pH, which confirms the assumption about the influence of the protein charge on the ability to penetrate the outer membrane (Fig. 3). The antibacterial activity of LysSt11 remained high over the entire pH range.

5 mM Sodium Phosphate Buffer (pH 7.5) is an excellent substitute for Tris containing buffer. it is more physiological in its composition. By adding various concentrations of alkali metal salts (NaCl and KCl), we evaluated their effect on the bactericidal activity of LysSi3 and LysSt11. It was shown that, starting from a concentration of 50 mM for NaCl and KCl, inhibition of the enzyme action is observed. Although at a concentration of 250 mM for both salts, the lytic effect of lysines is partially restored, reaching 60% of the initial level (Fig. 4).

The breadth of the spectrum of action was assessed on 16 strains of gram-negative bacteria of various genera and species and 2 strains of gram-positive bacteria of different origins: clinical isolates and laboratory strains (Table 1). It was shown that LysSi3 and St11 have a different bactericidal effect in relation to all studied strains, except for the gram-positive Staphylococcus aureus Z 73-14 (Fig. 5).

Table 1. Spectrum of antimicrobial activity of LysSi3 and St11 against gram-negative bacteria. + presence of an effect, - no effect.

Bacterial strain Antimicrobial activity LysSi3 LysSt11 Pseudomonas aeruginosa Ts 38-16 + + Pseudomonas aeruginosa Ts 43-16 + + Pseudomonas aeruginosa Ts 44-16 - + Acinetobacter baumannii Ts 50-16 + + Acinetobacter baumannii Ts 53-16 + + Acinetobacter baumannii Ts 58-16 + + Acinetobacter baumannii Ts 54-16 + + Klebsiella pneumoniae Ts 141-14 + + Klebsiella pneumoniae Ts 08-15 + + Klebsiella pneumoniae F 104-14 + + Klebsiella pneumoniae B3060 - - Klebsiella pneumoniae Osh-2k - - Klebsiella pneumoniae I6208 - - Escherichia coli M15 + + Salmonella typhi MVP 728 + + Staphylococcus aureus Z 73-14 - - Staphylococcus haemolyticus G 58-0916 - - Escherichia coli BL21 (DE3) pLysS - -

Thus, endolysin LysSi3 and LysSt11, without the addition of various permeabilizers or covalently bound penetrating peptides, is able to penetrate the outer membrane of gram-negative bacteria and degrade peptidoglycan, which ultimately leads to the death of bacteria. A significant decrease in the number of colonies grown on Petri dishes after incubation of bacteria and protein for a short amount of time indicates a high activity of LysSi3 and LysSt11. Thus, these endolysins are promising molecules for combating pathogens of the ESKAPE group.

Example 1. Obtaining strains of bacteriophages Salmonella typhi SI3 and S. typhi ST11 and their lysates.

For bacteriophage strains and Salmonella typhi SI3 ST11, active against Salmonella typhi, bacterial culture of the host strain at a titer of 10 ⁸ cfu / ml was inoculated into the culture vessel at a chamfered solid growth medium having a layer thickness of from 10 mm to 25 mm.

Each host strain was cultured for 3 hours at the optimum temperature for its growth (37 ° C), then a uterine bacteriophage in a titer of 10 ⁶ pfu / ml was inoculated onto the resulting bacterial culture lawn, the culture vessel (glass microbiological mattress) was hermetically sealed and cultured bacteriophage for 13 hours at an optimum temperature of 37 ° C and an air layer thickness above the surface of a dense nutrient medium from 25 mm to 40 mm.

To obtain phagolysate, the bacteriophage was suspended from the surface of the dense nutrient medium with physiological saline in an amount of 0.04 ml per 1 cm ^{2 of the} surface of the dense nutrient medium. The resulting liquid was aspirated into a sterile container, chloroform was added and kept for 30 minutes with continuous aeration. Then the solution was centrifuged for 30 minutes at 6000 rpm, the supernatant was sterilized by filtration through a filter with a pore diameter of 0.2 μm, and the resulting filtrate was passed through a column containing an agent affinity for endotoxin.

The resulting eluate was a purified phagolysate and contained bacteriophages in a titer of 10 ¹¹ PFU / ml in the absence of bacteria, ingredients of the used culture medium and at an endotoxin concentration of less than 50 units of endotoxin per 1 ml of purified phagolysate (EU / ml).

Example 2. Obtaining expression vectors carrying the coding sequences LysSi3 and LysSt11.

The coding sequences for endolysins LysSi3 and LysSt11 were obtained by PCR amplification using gene-specific primers (Table 2) from bacteriophage lysate. The obtained nucleotide sequences were placed in the pET42b (+) expression cassette. The correctness of the assembly of vector constructs was checked by the Sanger sequencing method. For purification of recombinant products using affinity chromatography, the coding sequences LysSi3 and LysSt11 were fused to the sequence coding for the 8-histidine tag at the C-terminus.

Table 2. Sequences of primers used to obtain the coding sequence for LysSi3 and St11.

LysSi3 Direct 5'-AAG AAG GAG ATA TAC ATA TGC AAC TCT CAA GAA AAG GT-3 ' LysSi3 Reverse 5'-TGG TGG TGG TGG TGC TCG AGC TTT GGG TAT ACA CTG TCA AGA TA-3 ' LysSt11 Straight 5'-AAG AAG GAG ATA TAC ATA TGC AAC TCT CAA GAA AAG GTT T-3 ' LysSt11 Reverse 5'-TGG TGG TGG TGG TGC TCG AGC TTT GGA TAT ACA CTG TCA AGA TAA-3 '

Thus, we obtained genetically engineered constructs based on the pET42b (+) vector encoding the LysSi3 and LysSt11 sequences for further expression of recombinant enzymes.

Example 3. Obtaining recombinant proteins LysSi3 and LysSt11 and their purification.

Expression vectors were placed in competent cells of E. coli strain BL21 (DE3) pLysS using a heat shock transformation protocol.

E. coli cells were grown in LB medium (37 ° C, 240 rpm) to OD600 = 0.55-0.65 and induction was performed with 1 mM IPTG at 37 ° C for 3 hours. The resulting cells were precipitated by centrifugation (6000 g, 10 min, at 4 ° C), resuspended in a buffer containing 20 mM Tris HCl pH 8.0, 250 mM NaCl, 0.1 mM EDTA), incubated with 100 μL / ml lysozyme at room temperature for 30 minutes and then destroyed by sonication. The resulting lysate was centrifuged at 10000 g for 30 minutes at 4 ° C, and then the supernatant was filtered through a filter with a pore size of 0.2 μm. The cleaning was carried out on an NGC Discovery ™ 10 chromatograph (Bio-Rad, USA) using a 5 ml HisTrap FF column (GE Healthcare, Germany) charged with Ni ²⁺ ions. The lysate was mixed with 30 mM imidazole and 1 mM MgCl ₂ and loaded onto the column, pre-equilibrated with a buffer containing 20 mM Tris HCl pH 8.0, 250 mM NaCl, 30 mM imidazole. Protein fractions were eluted from the sorbent using a linear gradient to 100% buffer containing 20 mM Tris HCl pH 8.0, 250 mM NaCl, 500 mM imidazole. The collected fractions were dialyzed against 20 mM Tris HCl pH 7.5 buffer. Protein purity was determined by electrophoresis 16% SDS-PAGE.

The protein concentration was determined spectrophotometrically at a wavelength of 280 nm (Implen NanoPhotometer, IMPLEN, Germany). The concentration was calculated taking into account the absorption coefficient E ^0.1% _280nm .

Thus, the recombinant endolysins LysSi3 and LysSt11 were obtained biotechnologically and prepared for further experiments to assess the bactericidal activity.

Example 4. Study of the antibacterial properties of LysSi3 and LysSt11.

An overnight culture of bacterial cells (OD600 = 1.2-1.4) was used as a culture of bacteria in the stationary growth phase or diluted 30 times with LB medium and grown to an exponential growth phase (OD600 = 0.6). The cells were pelleted by centrifugation (3000 g, 10 min) and resuspended in the same volume of 20 mM Tris HCl pH 7.5. The suspension was diluted 100-fold in the same buffer to a final density of about 10 ⁶ cells / ml. After 100 μl, the bacterial suspension was mixed with 100 μl of protein at the desired concentration in a 96-well plate. Buffer without added protein was used as negative control. The mixture was incubated at 37 ° C for 30 minutes (on a shaker 200 rpm) and then diluted 10 times with phosphate-buffered saline (pH 7.4). 100 μl of each dilution was applied to Petri dishes with LB agar medium. Colonies on the plates were counted after overnight incubation (18 hours) at 37 ° C. All experiments were carried out in three independent replicates.

Antibacterial activity was expressed as log10 of the number of colonies, or as the residual activity of LysSi3 and LysSt11 (%) compared to the control without the addition of enzymes.

In the course of experiments to study the antimicrobial properties of LysSi3 and LysSt11, it was found that the enzymes have a bactericidal effect on exponentially growing cells of the clinical strain A. baumannii Ts 50-16 at a concentration of 10 μg / ml (Fig. 1). However, when bacteria are exposed to the stationary growth phase, the bactericidal effect disappears, presumably due to a decrease in the ability of the protein to penetrate the outer membrane. In this case, with the addition of substances that increase membrane permeability (0.5 mM EDTA), the lytic effect is partially restored (Fig. 2).

Example 5. Study of the effect of pH, salts and buffers on the activity of LysSi3 and LysSt11.

The effect of pH, salts, and buffers (Tris HCl pH 7.5 and sodium or potassium phosphate buffer pH 7.5) on the specific activity of endolysins was assessed using the exponentially growing A. baumannii Ts 50-16 strain. To study the effect of pH, bacteria were incubated with each protein according to the above protocol (Example 3) in 20 mM Tris HCl buffer with different pH values (from 5.0 to 9.0). The effect of EDTA on bactericidal activity at different pH values was evaluated with the addition of 0.5 mM permeabilizer during incubation of the bacterial suspension with endolysin. When selecting the optimal physiological buffer for the action, 5, 10, 50 mM sodium or potassium phosphate buffers (pH 7.5) were studied. And to assess the effect of alkali metal salts on the protein in 5 mM sodium phosphate buffer (pH 7.5), various concentrations of NaCl or KCl salts (0, 10, 25, 50, 100, 150, 250 and 500 mM) were added.

The antibacterial activity was expressed as the residual activity of LysSi3 and LysSt11 (%) compared to the control without the addition of the enzyme.

It was shown that at different pH values weakly acidic, as well as neutral and slightly alkaline conditions are optimal for the enzyme action. At pH 6.5-7.0, a slight decrease in activity is observed, however, the addition of 0.5 mM EDTA eliminates the effect of pH (Fig. 3).

More physiological is 5 mM sodium phosphate buffer (pH 7.5). When various concentrations of NaCl and KCl were added to LysSi3 and LysSt11 in this buffer, starting from 50 mM for NaCl and KCl, inhibition of the enzyme action was observed. Although at a concentration of 250 mM for both salts, the lytic effect is partially restored (Fig. 4).

Example 6. Study of the spectrum of activity of LysSi3 and LysSt11.

The spectrum of enzyme activity was studied according to the above protocol in a buffer of 20 mM Tris HCl (Example 3) using 16 strains of gram-negative bacteria of various genera and species and 2 strains of gram-positive bacteria. All used strains were deposited in the collections of the National Research Center for Epidemiology and Microbiology named after Honorary Academician NF Gamaleya and the State Collection of Pathogenic Microorganisms and Cell Cultures (GKPM-Obolensk).

Table 3. Bacterial strains and clinical isolates used in the study and their source.

Bacterial strain Source Pseudomonas aeruginosa Ts 38-16 Patient's sputum, hospital strain Pseudomonas aeruginosa Ts 43-16 Wound discharge, hospital strain Pseudomonas aeruginosa Ts 44-16 Patient urine, hospital strain Acinetobacter baumannii Ts 50-16 Patient's sputum, hospital strain Acinetobacter baumannii Ts 53-16 Patient's sputum, hospital strain Acinetobacter baumannii Ts 58-16 Flushing from the infusion pump, hospital strain Acinetobacter baumannii Ts 54-16 Flushing from the infusion pump, hospital strain Klebsiella pneumoniae Ts 141-14 Patient urine, hospital strain Klebsiella pneumoniae Ts 08-15 Patient's sputum, hospital strain Klebsiella pneumoniae F 104-14 Patient's sputum, hospital strain Klebsiella pneumoniae B3060 CSF of the patient, hospital strain Klebsiella pneumoniae Osh-2k Patient blood, hospital strain Klebsiella pneumoniae I6208 Flushing from the patient's nasopharynx, hospital strain Escherichia coli M15 Laboratory strain Salmonella typhi MVP 728 Laboratory strain Staphylococcus aureus Z 73-14 Flushing from the patient's nasopharynx, hospital strain Staphylococcus haemolyticus G 58-0916 Patient's urethra, hospital strain Escherichia coli BL21 (DE3) pLysS Laboratory Expression Strain

The antibacterial activity was expressed as the residual activity of LysSi3 and LysSt11 (%) compared to the control without the addition of enzymes.

It was shown that LysSi3 and LysSt11 have a bactericidal effect against all studied gram-negative strains, except for Klebsiella pneumoniae B3060, Klebsiella pneumoniae Osh-2k, Klebsiella pneumoniae I6208, and also did not inhibit the growth of gram-positive strains. In addition, LysSi3 showed no activity against Pseudomonas aeruginosa strain Ts 44-16 and Escherichia coli BL21 (DE3) pLysS expression strain (Fig. 5).

Figure 1 shows the bactericidal activity of various concentrations of LysSi3 (A) and LysSt11 (B) against A. baumannii Ts 50-16 strain. As a control, bacterial cells incubated with buffer instead of protein were used. The number of surviving colonies after 18 hours of incubation of Petri dishes is expressed as a decrease in log10 CFU / ml compared to control. For all experiments, mean values of standard deviations are shown after three independent experiments. An asterisk (*) indicates a significant bactericidal effect.

Figure 2 shows the bactericidal activity of endolysins LysSi3 (A) and LysSt11 (B) at concentrations of 10 and 100 μg / ml against A. baumannii Ts 50-16 strain in exponential (Exp) and stationary (St) growth phases without and with the addition of 0.5 mM EDTA. The residual enzyme activity (%) is shown in comparison with the control. NS - there is no statistically significant difference from the control culture without the addition of endolysin (p> 0.05, Mann-Whitney test). For all experiments, mean values of standard deviations are shown after three independent experiments. An asterisk (*) indicates a significant bactericidal effect.

Figure 3 shows the effect of pH and 0.5 mM EDTA on the antimicrobial activity of LysSi3 (A) and LysSt11 (B) at a concentration of 1 μg / ml against A. baumannii Ts 50-16. The residual enzyme activity (%) is shown in comparison with the control. NS - there is no statistically significant difference from the control culture without the addition of endolysin (p> 0.05, Mann-Whitney test). For all experiments, mean values of standard deviations are shown after three independent experiments.

Figure 3 shows the effect of salts on the lytic activity of LysSi3 (A) and LysSt11 (B) at a concentration of 10 μg / ml against A. baumannii Ts 50-16 The residual enzyme activity (%) is shown in comparison with the control. NS - there is no statistically significant difference from the control culture without the addition of endolysin (p> 0.05, Mann-Whitney test). All other values are statistically significantly different from the control. For all experiments, mean values of standard deviations are shown after three independent experiments.

Figure 5 shows the bactericidal activity of LysSi3 (A) and LysSt11 (B) at a concentration of 50 μg / ml against various strains of gram-negative and gram-positive bacteria. The residual enzyme activity (%) is shown in comparison with the control. HA - no bactericidal activity was observed. All values were statistically significantly different from the control culture without endolysin addition (p <0.05, Mann-Whitney test). For all experiments, mean values of standard deviations are shown after three independent experiments.

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Sequence listing

<110> Antonova Natalia Petrovna, RU, Vasina Daria Vladimirovna, RU, Gintsburg Alexander Leonidovich, RU, Tkachuk Artem Petrovich, RU, Gushchin Vladimir Alekseevich, RU. (Antonova N.P., Vasina D.V., Gintsburg A.L., Tkachuk A.P., Guschin V.A.) <120> Recombinant proteins LYSSI3 And LYSST11, their derivatives, has an antibacterial effect, nucleic acids, the number of vectors encoding the proteins and their derivatives, expression producing strains of proteins and their derivatives, antibacterial compositions containing purified recombinant molecules or their derivatives in the pure form, but ALSO IN VARIOUS COMBINATIONS OF MIXTURES INCLUDING PHARMACEUTICALLY ACCEPTABLE CARRIERS AND / OR PERMEABILIZERS, AS WELL AS THEIR USE FOR ANTIMICROBIAL THERAPY <160> 8 <210> 1 <211> 154 <212> PRT <213> Bacteriophage SI3 <223> Amino acid sequence of bacteriophage SI3 endolysin <400> MQLSRKGLEA IKFFEGLKLE AYEDSAGIPT IGYGTIRIDG KPVKMGMKIT AEQAEQYLLA 60
DVEKFVAAVN KSIKVPTSQN EFDALVSETY NIGITAMQDS TFIKRHNAGN KVGCAEAMQW 120
WNKVTVKGKK VTSNGLKNRR RMEADIYLDS VYPK 154 <210> 2 <211> 154 <212> PRT <213> Bacteriophage ST11 <223> Amino acid sequence of ST11 bacteriophage endolysin <400> MQLSRKGLEA IKFFEGLKLE AYEDSAGIPT IGYGTIRIGG KPVKMGMKIT AEQAEQYLLA 60
DVEKFVAAVN KAIKVPTSQN EFDALVSETY NIGITAMQDS TFIKRHNAGN KVGCAEAMQW 120
WNKVTVKGKK VTSNGLKNRR RMEADIYLDS VYPK 154 <210> 3 <211> 465 <212> DNA <213> Bacteriophage SI3 <223> Nucleotide sequence encoding bacteriophage SI3 endolysin <400> ATGCAACTCT CAAGAAAAGG TTTAGAAGCT ATTAAGTTCT TTGAAGGTCT TAAGCTAGAG 60
GCTTACGAAG ATTCTGCCGG AATCCCAACA ATCGGGTATG GTACAATCCG TATTGACGGA 120
AAACCTGTTA AGATGGGTAT GAAAATTACG GCTGAACAAG CTGAACAGTA TCTTCTCGCA 180
GATGTTGAGA AGTTCGTTGC AGCGGTGAAC AAAGCCATCA AGGTTCCAAC TTCTCAGAAC 240
GAGTTCGATG CACTTGTAAG TGAAACATAC AACATCGGTA TCACAGCTAT GCAGGATTCT 300
ACATTTATCA AGCGCCACAA CGCTGGTAAT AAGGTAGGTT GTGCAGAAGC TATGCAGTGG 360
TGGAACAAGG TTACAGTCAA AGGTAAGAAG GTCACTTCAA ACGGCCTGAA AAACAGACGT 420
AGAATGGAAG CTGACATTTA TCTTGACAGT GTATACCCAA AGTAA 465 <210> 4 <211> 465 <212> DNA <213> Bacteriophage ST11 <223> Nucleotide sequence encoding bacteriophage ST11 endolysin <400> ATGCAACTCT CAAGAAAAGG TTTAGAAGCT ATTAAGTTCT TTGAAGGTCT TAAGCTAGAG 60
GCTTACGAAG ATTCTGCCGG AATCCCAACA ATCGGGTATG GGACAATCCG TATTGGTGGG 120
AAGCCTGTTA AGATGGGTAT GAAAATTACG GCTGAACAAG CTGAACAGTA TCTTCTCGCA 180
GATGTTGAGA AGTTCGTTGC AGCGGTGAAC AAAGCCATCA AGGTTCCAAC TTCTCAGAAC 240
GAGTTCGATG CACTTGTAAG TGAAACATAC AACATCGGTA TCACAGCTAT GCAGGATTCT 300
ACATTTATCA AGCGCCACAA CGCTGGTAAT AAGGTAGGTT GTGCAGAAGC TATGCAGTGG 360
TGGAACAAGG TTACAGTCAA AGGTAAGAAA GTCACTTCAA ACGGCCTGAA AAACAGACGT 420
AGAATGGAAG CTGACATTTA TCTTGACAGT GTATATCCAA AGTAA 465 <210> five <211> 164 <212> PRT <213> pET42b (+) LysSi3 <223> Amino acid sequence of recombinant endolysin LysSi3 containing an 8-histidine tag <400> MQLSRKGLEA IKFFEGLKLE AYEDSAGIPT IGYGTIRIDG KPVKMGMKIT AEQAEQYLLA 60
DVEKFVAAVN KSIKVPTSQN EFDALVSETY NIGITAMQDS TFIKRHNAGN KVGCAEAMQW 120
WNKVTVKGKK VTSNGLKNRR RMEADIYLDS VYPKLEHHHH HHHH 164 <210> 6 <211> 164 <212> PRT <213> pET42b (+) LysSt11 <223> Amino acid sequence of recombinant endolysin LysSt11 containing an 8-histidine tag <400> MQLSRKGLEA IKFFEGLKLE AYEDSAGIPT IGYGTIRIGG KPVKMGMKIT AEQAEQYLLA 60
DVEKFVAAVN KAIKVPTSQN EFDALVSETY NIGITAMQDS TFIKRHNAGN KVGCAEAMQW 120
WNKVTVKGKK VTSNGLKNRR RMEADIYLDS VYPKLEHHHH HHHH 164 <210> 7 <211> 495 <212> DNA <213> pET42b (+) LysSi3 <223> Nucleotide sequence encoding recombinant endolysin LysSi3 containing an 8-histidine tag <400> ATGCAACTCT CAAGAAAAGG TTTAGAAGCT ATTAAGTTCT TTGAAGGTCT TAAGCTAGAG 60
GCTTACGAAG ATTCTGCCGG AATCCCAACA ATCGGGTATG GTACAATCCG TATTGACGGA 120
AAACCTGTTA AGATGGGTAT GAAAATTACG GCTGAACAAG CTGAACAGTA TCTTCTCGCA 180
GATGTTGAAA AGTTCGTTGC AGCAGTGAAC AAATCCATCA AGGTTCCAAC TTCCCAGAAT 240
GAGTTTGACG CACTTGTAAG TGAAACATAC AACATTGGCA TCACAGCTAT GCAGGACTCT 300
ACATTTATCA AGCGCCACAA CGCTGGTAAT AAGGTAGGTT GTGCAGAAGC TATGCAGTGG 360
TGGAACAAGG TTACAGTCAA AGGTAAGAAA GTCACTTCAA ACGGCCTGAA AAACAGACGT 420
AGAATGGAAG CTGACATTTA TCTTGACAGT GTATACCCAA AGCTCGAGCA CCACCACCAC 480
CACCACCACC ACTAA 495 <210> 8 <211> 495 <212> DNA <213> pET42b (+) LysSt11 <223> Nucleotide sequence encoding recombinant endolysin LysSt11 containing an 8-histidine tag <400> ATGCAACTCT CAAGAAAAGG TTTAGAAGCT ATTAAGTTCT TTGAAGGTCT TAAGCTAGAG 60
GCTTACGAAG ATTCTGCTGG AATCCCTACA ATTGGGTATG GGACAATCCG TATTGGTGGG 120
AAGCCTGTTA AGATGGGTAT GAAAATTACG GCTGAACAAG CTGAACAGTA TCTTCTCGCA 180
GATGTTGAGA AGTTCGTTGC AGCGGTGAAC AAAGCCATCA AGGTTCCAAC TTCTCAGAAC 240
GAGTTCGATG CACTTGTAAG TGAAACATAC AACATCGGTA TCACAGCTAT GCAGGATTCT 300
ACATTTATCA AGCGCCACAA CGCTGGTAAT AAGGTAGGTT GTGCAGAAGC TATGCAGTGG 360
TGGAACAAGG TTACAGTCAA AGGTAAGAAG GTCACTTCAA ACGGCCTGAA AAACAGACGT 420
AGAATGGAAG CTGACATTTA TCTTGACAGT GTATATCCAA AGCTCGAGCA CCACCACCAC 480
CACCACCACC ACTAA 495

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Claims (4)

1. An antibacterial composition having an antibacterial effect against bacteria Acinetobacter baumannii, comprising an effective amount of the protein SEQ ID NO: 1 or SEQ ID NO: 5 and a pharmaceutically acceptable carrier. 2. An antibacterial composition having an antibacterial effect against bacteria Acinetobacter baumannii, comprising an effective amount of the protein SEQ ID NO: 1 or SEQ ID NO: 5, pharmaceutically acceptable carriers and permeabilizers selected from ethylenediaminetetraacetic acid (EDTA) and / or its salts. 3. The use of the protein SEQ ID NO: 1 or SEQ ID NO: 5 as an antimicrobial agent directed against bacteria Acinetobacter baumannii in its pure form. 4. Use of the protein SEQ ID NO: 1 or SEQ ID NO: 5 in combination with pharmaceutically acceptable carriers and / or membrane permeability enhancing agents as an antimicrobial agent against Acinetobacter baumannii bacteria.

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