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Биолюминесцентная детекция внешних воздействий физической и химической природы на бактериитезисы доклада
- Авторы: Ломакина Г.Ю., Угарова Н.Н.
- Сборник: Биоантиоксидант: материалы Х Международной конференции, посвященной 105-летию со дня рождения академика Н.М. Эмануэля
- Тезисы
- Год издания: 2020
- Издательство: РУДН
- Местоположение издательства: М.
- Первая страница: 49
- Последняя страница: 51
- Аннотация: Bioluminescent systems for biological processes detecting are increasingly used in scientific research. Bioluminescent imaging is a convenient non-invasive method based on recording the luminescence of cells or tissues in real time. Living cells are sensitive to external effects, so bioluminescent test systems based on them allow us to study in dynamics their effects on cell viability, division ability, metabolic activity, survival and adaptation. Living cell systems are effective for drug screening, evaluation of toxic effects, study of bacterial and viral pathogenesis, etc.E. coli BL21 (DE3) Codon Plus cells producing thermostable firefly luciferase L.mingrelica which catalyzes luciferin oxidation reactions in the presence of ATP and magnesium ions were used. Luciferase activity within cells was initiated by the penetration of luciferin into the cell at pH 5.0 when the luciferin molecule was not charged. Inside the cell, optimal conditions for the reaction are realized (ionic force, the presence of magnesium ions and ATP, as well as the pH of the cytoplasm), which corresponds to the optimal activity of luciferase. The level of extracellular luciferase was determined after the addition of the reaction mixture (luciferin and ATP in pH 7.8 solution) to the supernatant. The determination of ATP in the cell extracts was carried out with a firefly luciferase ATP reagent using potato apyrase, if necessary, to remove extracellular ATP.ATP is a sensitive indicator of the metabolic activity of living cells. The ATP level is a constant in metabolically active cells. Thus, E. coli BL21 (DE3) cells expressing active luciferase contain ATP in an amount of 2 · 10-18 mol/cell. Cell division leads to increase in the intracellular ATP level, which is proportional to the number of microorganisms. Cell integrity is evidenced by low extracellular ATP (2-5% of total ATP). The living cell responds to any stressful effects by changing the ATP content. As a result of cell death or destruction, the synthesis of ATP stops, and there is a drop in the level of intracellular ATP as a result of its disposal by cell components, in particular intracellular ATPase.The effect of antibiotics and temperature on this test system was studied. When incubating cells in the presence of polymyxin B and colistin, a decrease in ATP content was observed with an increase in antibiotic concentration, indicating a decrease in metabolic activity, destruction and cell death. There was a good correlation between ATP content and CFU determined by the plate count method. However, even at high antibiotic concentrations, a low residual ATP content was maintained, while the microbiological method yielded a negative result. Perhaps this indicates the presence of living cells that have lost their ability to divide at a given time.With heating (37-60 ° C) of the E. coli suspension, an increase in ATP level was found at the initial moment of time, followed by a smooth decrease, which indicates the course of complex intracellular adaptation processes - accelerated synthesis of ATP, increased cellular respiration under conditions of reduced oxygen level during heating and, possibly, inactivation of intracellular ATPases.The level of intracellular luciferase when exposed to an antibiotic was proportional to the number of cells that retained the integrity of the cell membrane. When the E. coli cell suspension was heat, the rates of denaturation of intracellular luciferase and cell death were close. Therefore, the enzyme activity correlated with the number of viable cells both at temperature exposure and when exposed to polymyxins. Under the action of polymyxin, the content of extracellular luciferase increased, indicating damage to the cell membrane and an increase in the availability of the enzyme for the substrate mixture. This indicates the possibility of using this system to study the rate of cell wall damage and mechanism under various stressors.Thus, the developed bioluminescent system based on living E. coli cells allows us to describe simultaneously several indicators characterizing the functioning of cells that change during external exposure - a change in metabolic activity, viability, division ability.
- Добавил в систему: Ломакина Галина Юрьевна