ИСТИНА

Staphylococcus aureus (Gram-positive microorganism) causes a large number of dangerous diseases (wound, respiratory and intestinal infections) of humans and animals. S. aureus rapidly develops resistance to a broad spectrum of antibiotics used in clinical medicine. All the above mentioned contribute to an intensive search for new ideology of treatment of patients with staphylococcal infections. The use of bacteriophage lytic enzymes (cell wall degrading proteins that are important to both the initial and terminal stages of the phage infection and lytic cycle) as antimicrobial agents is a promising alternative to conventional antibiotic therapy. Phage endolysins reveal high efficacy in destroying antibiotic-resistant pathogens with a mg quantities able to be produced in a recombinant form. To assess the possibility of practical use it is essential to study the activity and stability of enzymes under different conditions. Both activity and stability of enzymes is an important parameter when considering the possibility of using these active catalysts in medicine. The aim of the study was to carry out a physicochemical investigation of lysins of phages K, phi11, phi80α, 8161, 2638a. It contained the investigation of the lysins activity and stability in physiological conditions (presence of NaCl, human serum, human plasma); the study of temperature inactivation of the enzymes (4-50°C, 0.2-0.4 mg/ml enzyme, different pH values). The optimum storage conditions for lysins were found under which the enzymes retain their initial activity within a year (4°C). The loss of the enzyme activity may be associated with structural changes (secondary structure, tertiary structure). It is shown that the higher the stability of the enzyme, the less it changes the secondary or tertiary structure during the inactivation process. The main disadvantage of lysins as candidate therapeutics is their immunogenicity. The interaction between lysin (lysins of phages K and phi11) molecules and polymers (poly-L-lysine-polyethylene glycol block-copolymers, poly-L-glutamic acid-polyethylene glycol block-copolymers) results in the formation of protein-polyelectrolyte complexes (nanozymes). Complexing with block-copolymers produces a stabilizing effect on lysins due to structure ordering. Nanozymes of lysins of phages K and phi11 with block-copolymers have demonstrated sufficient stability at the temperature of storage (4°C). Lysin molecules when reacting with block-copolymers are incorporated in the particle cores. This process occurs through the formation of electrostatic contacts between charged sites of the enzyme molecules and oppositely charged fragments of molecules of block-copolymers. Polyethylene glycol chains of the molecules of block-copolymers are probably interacting with the aqueous phase (PEG-corona). The inclusion in a polymer matrix may result in a reduced immunogenicity of lysin which is a prerequisite for use as antimicrobial agent. The study was supported by Russian Scientific Foundation (grant number 14-13-00731).