Bioluminescent test systems based on firefly luciferase for studying stress effects on living cells

Abstract

The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly luciferase. The possibilities of using this reaction to study the influence of external effectors of a physical and chemical nature (temperature exposure, additions of drugs, membrane-active compounds, etc.) on living cells (prokaryotes and eukaryotes) are considered. Examples of the use of test systems based on living cells producing thermostable firefly luciferase for monitoring cellular homeostasis are given. The study of the kinetics of changes in the concentration of ATP and luciferase inside and outside cells made it possible to determine in dynamics the metabolic activity, cytotoxicity, and survival of cells under conditions of cellular stress, to study the processes of ATP synthesis/hydrolysis, and to evaluate the effectiveness of lytic agents in changing the permeability of the cell membrane.

Bioluminescence is convenient and sensitive instrument for investigation of stress effects on living cells due to its high sensitivity, specificity, and ability to rapidly record a bioluminescence signal (Fernández-Piñas et al. 2014; Syed and Anderson 2021). The advantages of bioluminescent methods over fluorescent ones are that they do not require external lighting, have extremely low background signals and are suitable for carrying out continuous long-term real-time monitoring in live cell cultures with a single cell resolution. Bioluminescence of firefly luciferin-luciferase system is of particular interest (Lomakina et al. 2015), because one of its substrates is ATP—an important indicator of cell viability. The oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions catalyzed by the firefly luciferase is accompanied by the emission of visible light. The intensity of the bioluminescent signal is proportional to the ATP and luciferase content in the sample

$$\begin{array}{c}\mathrm{D}-\mathrm{Luciferin}+{\mathrm{O}}_2+\mathrm{ATP}+{\mathrm{Mg}}^{+2}\\ \to \mathrm{Oxyluciferin}+\mathrm{AMP}+{\mathrm{PP}}_{\mathrm{i}}+\mathrm{h}\nu ({\mathrm{\lambda }}_{\max }\mathrm{at}\sim 560\mathrm{nm})\end{array}$$

This reaction is becoming more and more in demand for investigation of stress effects on living systems due to its excellent analytical characteristics—the high quantum yield, the unique specificity and selectivity of the reaction to ATP and luciferase, the high activity and stability of the bioluminescent signal and possibility of its application in non-invasive analysis of the cells in real-time, in vivo, and in situ. This reaction allows to determine ultra-low concentrations of two main components—ATP and luciferase in various biological samples with a limit of the detection ~ 10⁻¹² M below. For a long time, the low thermal stability of the firefly luciferase did not allow to use firefly luciferase in experiments with living cells at raised temperatures. The mutant luciferase with a high catalytic activity was obtained in our laboratory by directed evolution, that had a half-life of 10 h at 42 °C, which is 65-fold higher compared with the wild-type luciferase (Koksharov and Ugarova 2011). The ATP reagent based on this enzyme was developed (Ugarova et al. 2009). It contains all needed components to register a stable bioluminescence signal which is proportional to the ATP over a wide range of ATP concentrations (the correlation coefficient is 0.99, detection limit ~ 10⁻¹² M ATP) and it is suitable for repeated, long-term the ATP measurements in living systems.

ATP as an indicator of stress effects on living cells

All living organisms generate ATP through catabolic reactions and use it for providing vital activity of cells (Braissant et al. 2020; Ihssen et al. 2021). The intracellular concentration of ATP in actively growing cells is maintained at a constant level. For example, bacterial cells contain from 1 to 5 mM ATP and this distinction can be explained by the different size of the cells (Mempin et al. 2013; Bajerski et al. 2018). It is important to note that there is a linear relationship between the concentration of intracellular ATP and CFU, determined by the classical microbiological method. We used two approaches to control the concentration of the intracellular ATP (ATP_in).

The BCG vaccine, based on a live strain of Mycobacterium bovis BCG is widely used for the immunoprophylaxis of tuberculosis. One of the BCG vaccine’s key parameters is its cell viability (specific activity: the number of colony forming units, CFUs), which is traditionally defined by the microbiological method. The rapid and selective bioluminescent method of intracellular ATP assay (Ugarova et al. 2016) was used to control the BCG vaccine’s viability at various stages of the vaccine’s production. It permitted to reduce the time required for the analysis from 28 days to 1 h and to monitor the changes in metabolic activity of mycobacteria at all stages of the vaccine production. As the samples contained some concentration of extracellular ATP (ATP_ex), the assay protocol included the steps shown below (Ugarova and Lomakina 2017):

1. Degradation of ATP_ex by incubation of the sample with apyrase for 10 min (Lomakina et al. 2020a, b);

2. Lysis of cells by dimethyl sulfoxide for 1 min to release intracellular ATP into solution (Lomakina et al. 2015);

3. Measurement of intracellular ATP concentration using bioluminescent ATP reagent (Ugarova et al. 2009).

The correlation was shown between the viability of a liquid BCG vaccine measured by the microbiological method compared to one calculated using the content of intracellular ATP, as well as the correlation between the CFU value for the lyophilized BCG vaccine and the ATP content in the liquid vaccine before lyophilization. Consequently, after measuring the ATP in the liquid vaccine specimens before lyophilization, one can predict the cells’ viability in final lyophilized product. Thus, the operational ATP assay ensures control of the cells’ viability of the BCG vaccine during the process of its production, which is necessary in order to provide a high quality of the produced drug manufactures (Ugarova et al. 2019).

Another approach was used in the study of the kinetics of the interaction of E. coli cells with the membrane-active antibiotic colistin. In this case, it was necessary to measure the dynamics of changes in the concentration of ATP outside and inside the cells in the presence of colistin.

The protocol included the following steps (Lomakina and Ugarova 2022a, b):

1. Measurement of the ATP concentration outside the cells (ATP_ex) adding the ATP reagent to the cell suspension at pH 7.8, when neither luciferase nor luciferin penetrate E. coli cells;

2. Lysis of cells by dimethyl sulfoxide for 1 min to release intracellular ATP into solution;

3. Measurement of total ATP (ATP_tot) concentration (the sum of extracellular (ATP_ex) and intracellular (ATP_in) ATP concentrations);

4. Calculation ATP_in by the difference of ATP_tot and ATP_ex.

The study of dynamics of changes in the ATP concentration outside and inside the cells in the presence of various concentrations of the antimicrobial agents gave an additional information on the mechanism of their action. The ATP_ex level serves as an indicator of changes in the permeability of the cell membrane under the action of various agents causing the leakage of intracellular ATP into the extracellular space. The applicability of this test system has been demonstrated by the example of a membrane-active agent, the antibiotic polymyxin E (colistin).

Polymyxins have been known for about 80 years, nevertheless, the mechanism of their action has been widely discussed in recent time. The action of membrane-active antibiotics of colistin was investigated on gram-negative pathogen—E.coli cells (Lomakina and Ugarova 2022b). The first target of these cationic polypeptides is the outer membrane of microbial cells (Moubareck 2020). It was shown that only slight increase in leaked ATP_ex was observed upon incubation of the cell suspension with colistin. The most of the ATP from the initial pool of ATP_in is not detected either outside or inside the cells and the rate of decrease in intracellular ATP concentration significantly exceeded the rate of accumulation of extracellular ATP concentration. It could be assumed that a violation of the permeability of the outer membrane of cells during incubation with colistin should lead to the release into the extracellular space of not only ATP, but also enzymes that utilize ATP. The absence of hydrolysis of ATP_ex additionally introduced into the reaction mixture indicated that in the extracellular space, ATPases from E. coli cells do not function. Thus, ATP is utilized inside the cell and the rate of its consumption is not compensated by the rate of the ATP synthesis. There is the imbalance between the processes of synthesis/hydrolysis of ATP inside the cell. As a result, we observed a sharp decrease in the concentration of ATP_in which is probably due to a decrease in the activity of the respiratory chain enzymes and ATP synthase which operate in the cytoplasmic cell membrane (Sabnis et al. 2021). It leads to a decrease in the rate of ATP synthesis or even to its halt. So, we conclude that polymyxin could probably not only disrupt the cell membrane, leading to the release of intracellular components, but also affect a number of other intracellular processes such as the respiratory chain enzymes and ATP synthase. Comparison of the dynamics of ATP changes in cells incubated in culture medium and in saline demonstrated that non-growing cells were more resistant to the action of colistin. The decrease in ATP level decreases the activity of antibiotic targets, which may be a first step towards the formation of persisters (Shan et al. 2017), a subpopulation of cells tolerant to the antibiotic action and causing chronic infections.

Luciferase as indicator of the cell permeability

Using recombinant cells producing firefly luciferase, we showed that firefly luciferase is an informative protein marker for study changes in cell membrane permeability under the action of membrane active agents since the synthesis of the enzyme in the cell does not depend on the activity of enzymes functioning in the cell membrane, which is one of the targets of these agents action (Lomakina and Ugarova 2022a). Luciferase activity inside (A_in) and outside (A_ex) Escherichia coli (strain BL-21 (DE3) codon plus) bioluminescent cells, which produce thermostable luciferase of fireflies Luciola mingrelica was measured 1 h after the start of incubation in the absence and presence of different concentrations of colistin, as it is shown at Fig. 1.

Fig. 1.

Interaction of colistin with E. coli cells

The value of A_in decreased with increasing colistin concentration up to background values at 0.2 mg/mL colistin. At the same time, the value of A_ex increased rapidly with increasing colistin concentration. Thus, an increased A_ex value indicated the occurrence of severe defects in the cell wall. Partial or complete destruction of the cell membrane may cause endogenous luciferase to leak into the solution, resulting in an increase in A_ex value. It was shown that colistin does not affect the enzymatic activity of luciferase. Luciferase activity can be determined very rapidly and with very high sensitivity. All these make firefly luciferase a promising marker for the study of cell membrane permeability (Lomakina and Ugarova 2022a).

Firefly luciferase was also applied as reporter for investigation of stress effect of membrane active saponins—digitonin and its analogs on the eucariotic cells. Digitonin is widely used for the preparation of medical compositions to increase their permeability into pathogenic cells. It is known that digitonin forms complexes with cholesterol of cell membranes which leads to a change in permeability of the membrane structure, the formation of pores, and the release of intracellular components. Unfortunately, this effector is used at a fixed concentration and incubation time and its lytic effect is evaluated at two points—before and after addition of agent. HEK293 cells transiently transfected with the pcDNALuc plasmid expressing firefly luciferase were used for studying of the early stage of the interaction of cell membranes with digitonin (Lomakina et al. 2020a). In real time, the kinetic curves of accumulation of luciferase activity in supernatant during incubation of different digitonin concentrations with cells were recorded at pH 7.8 in the presence of luciferin which does not penetrate into the native cells. The high molecular weight enzyme becomes available to lucifetrin only with significant damage to the cell membrane. It was shown that lytic effect on cells depended on the digitonin concentration. At a concentration of less than 0.02 mM, a long induction period was observed, when the luciferase activity outside the cells did not registered. With an increase in the concentration of digitonin to 0.04 mM, the induction period was shortened and the concentration of released luciferase increased rapidly, which indicates the formation of large pores. With an increase in the concentration of digitonin from 0.04 to 0.2 mM the rate of luciferase release increased by 64 times. Digitonin was the most toxic to cells at the concentration ≥ 0.08 mM (Lomakina et al. 2020a). The influence of saponin structure on their lytic activity also has been studied. A decrease in a number of carbohydrate cycles in molecule of dioscin (analog of digitonin) leads to a decrease in its lytic activity. The presence of a bulky substituent in the aglyconic part of the molecule protodioscin (another analog of digitonin) prevents binding to the membrane (Lomakina et al. 2020a). Thus, bioluminescence method offered to study in situ changes in the permeability of cell membranes under the action of membrane-active effectors in the early stages of cell lysis.

Endogenic TSLuc as an indicator of temperature stress on living cells

The search for sensitive, easily, and quickly detectable endogenous indicators to study the effect of elevated temperatures on living cells is of great scientific and practical importance, in particular, to ensure microbiological safety when optimizing food processing conditions (Smelt and Brul 2014). Thermostable firefly luciferase produced by recombinant E. coli BL21 cells was shown to be a sensitive indicator for studying the effect of temperature on the viability of living cells (Lomakina et al. 2018). The bioluminescent signal is high and long. It can be registered for several minutes without a significant decrease in intensity. Luciferase is expressed in a soluble and active form in the cell, which makes it possible to measure its enzymatic activity directly inside the cells without destroying it (Fig. 2). Kinetic parameters of thermal activation of luciferase in E. coli cells turned out to be similar to the parameters for the purified enzyme in the buffer system. In both cases, enzyme inactivation obeys first-order kinetics regardless of the microenvironment. Kinetics of the thermal inactivation of intracellular luciferase was studied by bioluminescent method. In parallel, kinetics of the cell viability decrease was obtained by the seeding method. The rate constants of both processes were practical identical (Table 1).

Fig. 2.

Measurement of luciferase activity inside (A_in) and outside (A_ex) Escherichia coli cells

Table 1.

The rate constants of inactivation of luciferase and the rate constants of the cell viability decrease for recombinant E. coli cells (strain BL-21 (DE3) codon plus) at different temperatures

	Endogenic luciferase	E. coli (CFU)
t, oC	kin, min−1	kin, min−1
45	0.012 ± 0.001	0.014 ± 0.003
47	0.028 ± 0.002	0.024 ± 0.005
50	0.133 ± 0.007	0.121 ± 0.01
55	1.2 ± 0.03	0.97 ± 0.11

Temperature-induced cell death (loss of viability) has been shown to be due to the denaturation of a subset of intracellular proteins with key functions (Leuenberger et al. 2017). The found correlation between the rate constants of inactivation of endogenous luciferase and the decrease in the viability of E coli cells (Table 1) indicates that firefly luciferase is indeed a highly informative indicator when studying the temperature stress of bacteria. The advantage of luciferase is the possibility of simple and rapid detection of its activity by the bioluminescent method in comparison with the classical microbiological method of the cell viability assay.

Conclusion

Two bioluminescent intracellular indicators—low-molecular weight ATP and high-molecular weight thermostable firefly luciferase—were used for studying of cell response to stressors, monitoring of cellular homeostasis and molecular changes in time. They were shown to be highly informative for study the mechanisms of cell adaptation, survival, and cell death in the presence of external effectors of a physical and chemical nature (temperature, drugs, membrane-active compounds, etc.) on living cells (prokaryotes and eukaryotes). The investigation of the kinetics of changes in the ATP concentration and luciferase activity inside and outside cells made it possible to determine in dynamics the processes ATP synthesis/hydrolysis and the cell membrane permeability under the action of the stress agents.

For many years, bioluminescent methods have been widely used to study the effects of chemical and physical agents on living organisms. Most of these studies are done using glowing bacteria and various marine organisms. Many interesting results have been obtained, presented in numerous experimental articles and reviews. Classification of physical and physico-chemical mechanisms of action of the exogenic agents (fluorescent dyes, organic oxidizers, organic and inorganic heavy-atom containing compounds, and metallic salts) on luminous bacteria Photobacterium leiognathi was suggested (Kudryasheva 2006). The effects of alpha- and beta-emitting radionuclides on marine microorganisms under conditions of chronic low-dose irradiation in aqueous media were studied. Three successive stages in the bioluminescent response to americium-241 and tritium were found: (1) absence of effects (stress recognition), (2) activation (adaptive response), and (3) inhibition (suppression of physiological function, i.e., radiation toxicity) (Kudryasheva and Rozhko 2015). The impacts of radioactivity of different types (alpha, beta, and gamma) and bioactive compounds (humic substances and fullerenols) were considered. Bioassays based on luminous marine bacteria, their enzymes, and fluorescent coelenteramide-containing proteins were used to compare the results of the low-intensity exposures at the cellular, biochemical, and physicochemical levels, respectively (Kudryasheva and Kovel 2019).

This review presents another new direction in the use of bioluminescence as a highly sensitive indicator for studying stress effects on living organisms.

Funding

This work was performed as part of the state registration topic of Lomonosov Moscow State University, no. 121041500039–8

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Informed consent was obtained from all authors of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.