Relationship between the equilibrium parameters of lysozyme adsorption on the surface of living cells of bacteria of various bacterial families and the kinetics of enzymatic lysis of bacterial cells on the example of E. coli, B. subtilis, L. plantarum

Levashov, P.A.; Rastriga, N.V.; Smirnov, S.A.

Авторы: Levashov P.A., Rastriga N.V., Smirnov S.A.
Сборник: The 7th Congress of Biophysicists of Russia - conference proceedings, Biophys Rev, 15, 2023, 1425–1861, Biophys Rev, 15, 2023, 1425–1861
Серия: The 7th Congress of Biophysicists of Russia - conference proceedings
Том: 15
Год издания: 2023
Место издания: Springer Science Германия
Первая страница: 46
Последняя страница: 46
Номер статьи: S1.89
DOI: 10.1007/s12551-023-01150-w
Аннотация: For complex polymeric substrates, the importance of the type of enzyme adsorption has long been known. It can be either productive, i.e., preceding the catalytic stage of the process, or unproductive, in which it blocks the action of the enzyme. During the past two decades, new aspects have been discovered in association with a special form of bacterial resistance with respect to the action of antibacterial factors of the immune system. Increasingly, new molecular structures of the surface of bacterial cells are being revealed to be capable of purposely binding the antibacterial enzyme lysozyme, thereby blocking the mechanisms of bacterial disintegration during phagocytosis by an immune cell, which in turn blocks the process of neutralizing the bacterium and the subsequent presentation of bacterial antigens, the latter of which is necessary for the formation of specifc antibodies.In this study we examined the parameters of adsorption of the enzyme lysozyme directly on living whole bacterial cells under conditions of enzymatic lysis (destruction) of bacteria. Chicken-egg lysozyme, which is currently used in medical preparations and has a structure similar to that of human lysozyme, was used as a model enzyme. Gram-negative bacteria Escherichia coli of the Enterobacteriaceae family, Gram-positive spore-forming bacteria Bacillus subtilis of the Bacillaceae family, and Gram-positive Lactobacillus plantarum of the Lactobacillaceae family were studied as bacteria.It was shown that the adsorption of lysozyme is reversible and that equilibrium is established in less than three to fve minutes at a temperature of 37°C. Once equilibrium is established, the adsorption isotherms in all cases are satisfactorily approximated by the Langmuir equation. An analysis of the experimental data did not reveal the simultaneous presence of diferent lysozyme binding sites on bacterial cells, thus indicating that the equilibrium adsorption parameters difer signifcantly. With a diferent proportion of lysed cells, following incubations of a mixture of bacteria with lysozyme for 5 minutes and 20 minutes, respectively, the adsorption characteristics did not change in excess of the error margin, indicating that the nature of lysozyme adsorption on whole cells and on cell fragments is the same. The adsorption capacity is the maximum amount of lysozyme that can bind to bacterial cells under specifed conditions (Amax). Amax, however, decreases as the pH value rises and falls from the activity optimum. Amax ranges from 2.7·10^7 to 1.5·10^8 lysozyme molecules per cell, depending on the type of bacteria. In the case of bacterial cells E.coli and B.subtilis, a twofold decrease in the rate of enzymatic cell lysis (VL) was found along with a decrease in the ionic strength of the solution in the range of 80 mM to 5 mM. The study of kinetic parameters showed that a decrease in the rate of cell lysis occurs due to an increase in the Michaelis constant (Km) but that the maximum rate of cell lysis (Vmax) remains unchanged. It was found that, with a decrease in ionic strength, the desorption constant of lysozyme (Kd) decreases while Amax remains unchanged. Kd decreases up to 10 times for E. coli (upto 1.2·10^-7 M), and Kd decreases up to three times for B. subtilis (upto 2.4·10^-7 M). An increase in the contribution of unproductive adsorption of lysozyme on bacteria E. coli and B. subtilis can be assumed but with decreasing ionic strength. In the case of L. plantarum cells, when the ionic strength of the solution changes in the range of 80 mM to 5 mM, there is no signifcant change in the adsorption parameters of lysozyme and VL. In the case of E. coli, an increase in VL by a factor of 1.5 to 2.0 is observed in the presence of free amino acids glycine, histidine and charged amino acids (Gly, His, Asp, Glu, Arg, Lys) at a concentration of 1–5 mM. In the presence of these amino acids, Amax does not change, but Kd decreases. Consequently, the additives that activate enzymatic lysis probably increase the contribution of productive sorption.Bacterial infections of the Enterobacteriaceae and Bacillaceae families (e.g., typhoid fever, salmonellosis, bacillary dysentery, anthrax) are especially dangerous because these bacteria can bind the phagocyte lysozyme and use phagocytes as containers for spreading the bacteria throughout the body. Therefore, new information on efectors that prevent unproductive adsorption of lysozyme is particularly important in the development of new antibacterial drugs.The work was performed at the Department of Chemical Enzymology of the Faculty of Chemistry of Lomonosov Moscow State University within the framework of the state task “Molecular design, structural and functional analysis and regulation of enzyme systems, cellular structures, bionanomaterials: fundamental foundations and applications in technology, medicine, environmental protection” Number CITIS: 121041500039-8.
Добавил в систему: Левашов Павел Андреевич

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Relationship between the equilibrium parameters of lysozyme adsorption on the surface of living cells of bacteria of various bacterial families and the kinetics of enzymatic lysis of bacterial cells on the example of E. coli, B. subtilis, L. plantarumстатья

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