Bioelectromagnetics 35:136^144 (2014)
Electric Field-Induced Effects on Yeast Cell Wall Permeabilization Arunas Stirke,1,2* Aurelijus Zimkus,1,3 Almira Ramanaviciene,2 Saulius Balevicius,1 Nerija Zurauskiene,1 Gintautas Saulis,1 Larisa Chaustova,3 Voitech Stankevic,1 and Arunas Ramanavicius1,2 1 Center for Physical Sciences and Technology,Vilnius, Lithuania Center of Nanotechnology and Materials ScienceNanoTechnas, Faculty of Chemistry, Vilnius University,Vilnius, Lithuania 3 Department of Biochemistry and Biophysics,Vilnius University,Vilnius, Lithuania
2
The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPPþ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 ms and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPPþ absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPPþ by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPPþ absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPPþ, is due to the poration of the plasma membrane. The applicability of the TPPþ absorption measurements for the analysis of yeast cells electroporation process is also discussed. Bioelectromagnetics 35:136–144, 2014. © 2013 Wiley Periodicals, Inc. Key words: electroporation; tetraphenylphosphonium; pulsed electric field; yeast
INTRODUCTION For several decades, the budding yeasts (Saccharomyces cerevisiae) have been considered prototypical of eukaryotic cells, ideally suited for use in studies of many of the basic phenomena of eukaryotic life and some of their fundamental properties [Neumann et al., 1996; Breitenbach et al., 2012]. Yeasts are used not only to produce transgenic proteins, or for fermentation in foods and beverages, but also can be used as microbial fuels cells for generating green electricity [Ramanavicius and Ramanaviciene, 2009; Liu et al., 2011]. The S. cerevisiae yeast cells are surrounded by cell walls, which provide them with protection from osmotic stress and are also important for their defense against toxic compounds, for self-recognition and for flocculation [Aouida et al., 2003; Veelders et al., 2010; Levin, 2011]. Yeast cell walls are mainly composed of fibrous polysaccharide b-1,3-glucan and mannoproteins (proteins highly N-and O-glycosylated with mannose residues), which are located on the external 2013 Wiley Periodicals, Inc.
layers of the walls. Such mannosyl side chains are phosphorylated and provide an anionic surface charge to the yeast cell wall surfaces at physiological pH values [Jigami and Odani, 1999; Van Holle et al., 2012]. The cell wall hydrophobicity and charge are the major determinants of yeast adhesion on abiotic surfaces. Both play major roles in medical and bioprocessing industries as they form biofilms on human tissue and biofouling surfaces when used in the Grant sponsor: Lithuania Agency for Science, Innovation and Technology; grant number: 31V-33. *Correspondence to: Arunas Stirke, Center for Physical Sciences and Technology, A. Gostauto 11, LT-01108, Vilnius, Lithuania. E-mail: [email protected] Received for review 18 February 2013; Accepted 19 September 2013 DOI: 10.1002/bem.21824 Published online 6 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
PEF-Induced Yeast Cell Wall Permeabilization
food or beverage industries [Rhymes and Smart, 2001; White and Walker, 2011]. Several methods have been used to change the permeability of the yeast cells. These include treatment of the cells chemically by adding reducing agents such as dithiothreitol (DTT) or antibiotics [Brajtburg et al., 1990; Klis et al., 2007]. Their permeability can also be changed by increasing the external mechanical pressure on the cells [Smith et al., 2000], or by treating them with pulsed electric fields (PEFs; electropermeabilization or electroporation), which facilitates the leakage from the cells of intracellular compounds, such as proteins, ions, and nucleic acids [Liu et al., 2011]. In order to detect changes of the permeability of the yeast cells, fluorescent probes are used. Various charged molecule adsorption measurements are taken to determine the changes induced in the yeast cell wall surface charge by the PEF [Tomov and Tsoneva, 1989; Neumann et al., 1996]. Bleomycin has also been used as a probe in yeast viability studies affected by PEF. This enables analysis of the contributions of the cell wall and the cell membrane in limiting the uptake of a drug [Aouida et al., 2003]. Bleomycin is an antitumor drug, a hydrophilic molecule that is unable to cross the cell membrane by free diffusion, but this drug also has a positively charged tail [Chen and Stubbe, 2005]. For this reason, the molecule can be retained on the surface of the yeast cell. Other researchers have used indirect methods (electric conductivity of the medium and zpotential of yeasts cells) to analyze permeabilization and have proposed some hypothetical assumptions on the mechanism of the surface charge changes of yeast cells affected by PEF [El Zakhem et al., 2006a]. However, the quantitative measurement of the cell permeabilization using these methods is difficult. Moreover, only few methods can be used for the indication of selective cell wall permeabilization without causing lethal influences to the cells. One of the possible ways of increasing this selectivity is the application of lipophilic cations (LCs) as a probe for the indication of the permeability of the yeast cell walls. Tetraphenylphosphonium salts (e.g., tetraphenylphosphonium bromide [TPPBr]) are frequently used in the measuring the membrane potential (Dc) of prokaryotic and eukaryotic cells. The use of a potentiometric ion-selective electrode is also a convenient method for the quantitative evaluation of the permeability of the yeast cell wall and membrane [Zimkus and Chaustova, 2004]. The purpose of this study was to investigate the permeability of viable yeast cells after exposure to PEF using the indirect TPPþ selective probe-based method.
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MATERIALS AND METHODS Yeast Strain and Cultivation S. cerevisiae SEY6210 (MATa, leu2-3, leu2-112, ura3-52, his3-D200, trp1-D901, lys2-801, suc2-D9, GAL) yeast cells (donated by Prof. H. Bussey, McGill University, Quebec, Canada) were grown in a 250 ml of complete medium of yeast extract-peptone-dextrose (YPD; 1% yeast extract, 2% peptone, 2% glucose, Sigma–Aldrich, St. Louis, MO) at 30 8C in a reciprocal shaker operating at 150 rpm. Cell density was measured by optical density (OD) using an optical absorption spectrophotometer (Biophotometer; Eppendorf, Hamburg, Germany) operating at a wavelength of 600 nm. In order to collect the cell more sensitive to the physical treatments [Anton-Leberre et al., 2010] and to ensure the repeatability of results, the yeast cells were centrifuged when the OD achieved 0.8–1.0 for light path 1 cm. At the early exponential growth phase yeast cells were washed with distilled water and then resuspended in 1 ml of cold (4 8C) electroporation buffer (EPB) containing 1 mol/dm3 sorbitol 20 mmol/ dm3 Tris–HCl buffer, pH 7.4 (Sigma–Aldrich) keeping the final concentration of the yeasts at (4–6) 109 colony forming unit (CFU)/ml [Zimkus et al., 2006]. Electroporation Setup The experimental setup for the electroporation of the yeast is presented in Figure 1. It consisted of a high voltage DC power supply (HV source) ALE 500A (TDK-Lambda Americas, Neptune, NJ), a low voltage, microsecond square-wave pulse generator TG 5011 (Thurlby Thandar Instruments, Cambridgeshire, UK), a high voltage electrical pulse forming circuit (Fig. 1, shaded area), a real time oscilloscope DPO4034 (Tektronix, Beaverton, OR) and a commercial cuvette (Lonza, Cologne, Germany). The variable HV source was used to charge a 120 mF capacitor C up to a voltage from several volts to 1 kV. The charging of capacitors was performed through the charging resistor RCh ¼ 100 kV. The pulse generator creates square-wave pulses that were used for control of an isolated bipolar transistor (IGBT) 1MBH25D120 (Fuji Electric, Tokyo, Japan). During the experiments, pulse duration was varied from 0.1 ms to 1 ms. The circuit operated as follows: after charging the capacitor bank C, the square-wave pulse generator creates a required duration trigger pulse, which triggers the IGBT through a HCPL3120 gate driver (Avago Technologies, San Jose, CA). The IGBT was capable of withstanding collector-emitter voltages up to 1200 V and current pulses of lasting 1 ms with maximal amplitudes up to 114 A. The input circuit of the gate driver was optically isolated. A low Bioelectromagnetics
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Fig.1. The experimental setup used for yeast cell electroporation. The shaded area is a high voltage electrical pulse forming circuit which consists of the isolated gate bipolar transistor (IGBT), gate driver of this transistor, capacitor (C), ballast resistor (RB), capacitors charging resistor (RCh), and shunt resistor (RSh).
inductance ballast resistor RB ¼ 20 V was used to protect IGBT transistor from current overshoot. When this IGBT was switched on, the capacitor discharged a cuvette through the load with a buffer containing the yeast and ballast resistor. A real time oscilloscope connected in parallel to the shunt resistor RSh ¼ 2 V was used for the measurement and calculation of the pulse parameters: duration and waveform of current through the cuvette and electric field strength. The typical waveform of the measured pulse (VOsc) is presented in the inset of Figure 1. The rise time was 0.1 ms and the fall time was approximately 0.15 ms. It has to be noted that the shape of this waveform represents current through the cuvette (voltage measured across shunt resistor). Good square shape indicates that conductivity of the buffer with the yeast does not change during the pulse (no heating, electrolysis or other processes are observed during the pulse). The strength of the PEF Ep across the cuvette was calculated according to the equation Ep ¼ Vp/d; where d is the distance between the electrodes; Vp is the voltage drop across the cuvette: V p ¼ V S ðV Osc =RSh ÞðRSh þ RB Þ, where the VS is the power source voltage. A commercial cuvette 120 ml of total volume with plate-shape electrodes of 2 cm2 and gap between electrodes of 1 mm was used. Bioelectromagnetics
TPPþ Accumulation Analysis All electroporation procedures were carried out in abovementioned commercial cuvette filled with 120 ml of yeast suspension prepared in EPB. TPPBr to a final concentration of 5 106 mol/dm3 was added before the electroporation. After electroporation and incubation for an additional 3 min, the test samples were precipitated and TPPþ absorption measurements were performed in separated tubes by mixing 50 ml of resultant supernatant with 200 ml of electrode calibration solution (TPPþ concentration 2 106 mol/dm3). After that initial TPPþ concentration Ns was [TPPþ]ini6 mol/dm3. Such solution was used for the tial ¼ 2.6 10 measurement of the residual TPPþ ion concentration in supernatant ([TPPþ]supernatant). The quantity (N) of the TPPþ absorbed by the yeast cells was calculated as follows: N ¼ [TPPþ]initial [TPPþ]supernatant, that is, N ¼ Ns Nsupernatant. The control sample was prepared and measured at 0 kV/cm as well as test samples, which were treated using single square shape electric field pulses of 5, 10, 50, 100, and 150 ms duration and different electric field strengths up to 10 kV/cm. A combined tetraphenylphosphonium selective electrode was used for this purpose. This electrode has been described previously [Zimkus and Chaustova, 2004]. The electrode potential drift was estimated using a WTW 526 ionometer (WTW, Weilheim, Germany) connected to a PC. In order to investigate the TPPþ accumulation level, these measurements were conducted at different time intervals after electroporation and incubation of the EPB with TPPþ at room temperature (20 8C). Evaluation of the Proliferation of Yeast Cells After the Action of PEF The determination of the yeast cell viability was conducted before and after the electroporation procedure. The yeast proliferation assay was conducted using a microbial cell proliferation kit based on absorption changes of 2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8; Dojindo, Kumamoto, Japan) after an action of viable yeast cells. Two different mediums were used: an EPB buffer and a synthetic complete (SC) medium (colorless yeast nitrogen base medium; Sigma–Aldrich) with 2% of glucose (Merck, Darmstadt, Germany). After incubation of the yeast cells for 4 h at 30 8C, measurements of the absorption at a 460 nm wavelength were conducted with a Tecan Sunrise microplate reader (Tecan Group, Männedorf, Switzerland). The obtained results of the three experiments are presented as means with standard deviation.
PEF-Induced Yeast Cell Wall Permeabilization
The Dye Release Assay To determine the effectiveness of the plasma membrane electroporation, a calcein blue release assay was performed. The yeast cells were concentrated to 1.5 108 CFU/ml in 100 ml of the EPB buffer and then 400 mmol/dm3 of acetoxymethyl (AM) C1429 esterified calcein blue (Life Technologies, Eugene, OR), a cell permeable derivate of calcein blue, was added. After 30 min of incubation at room temperature (20 8C) and avoiding any exposure to light, the cells were observed using a BX51TF fluorescence microscope (Olympus, Tokyo, Japan) equipped with an appropriate filter cube. Images of the yeast cells were recorded using an Evolution QEi Monochrome camera (Media Cybernetics, Silver Spring, MD) and were processed using a Pro Express v.6 (Media Cybernetics) program. Measuring the percentages of fluorescent and non-fluorescent yeast cells in the cuvette using the dye release test determined the electroporation efficiency of the PEF. This test is based on the fact that the electroporated cells are practically permeable to such dyes as calcein. At least 100 yeast cells were counted in each experiment. The experiments were repeated three times. RESULTS The Accumulation of TPPþ by the Yeast Cells After Exposure to PEF The quantity of the TPPþ absorbed by the yeast cells was expressed as accumulation level and was given by the ratio (N/Nm) 100%. Here Nm is the maximal concentration of the molecules that can be accumulated inside the yeasts (steady-state accumulation level). Initial concentration of TPPþ (Ns) was always kept higher than Nm. It was determined that the concentration of the TPPþ accumulated in the yeasts cells depends on the incubation time of the cells with the LCs. For the various yeast strains, a steady-state accumulation level Nm is reached after a few hours of treatment in the buffer solutions [Zimkus and Chaustova, 2003]. The rate of the LC absorption of the untreated yeast cells is lower in comparison to the ones treated by the PEF. A TPPþ accumulation level of 90% was reached after 100 min for the untreated cells and after 40 min for the yeast cells treated with a 4.8-kV/cm electric field strength, 150 ms duration pulse (see Fig. 2, symbols). It has to be noted that the steady-state accumulation level is not changed by the PEF action on the yeast cells. The results of the TPPþ accumulation by the yeast cells that were obtained after treating them with a single square shape pulses having different
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Fig. 2. The experimental data (symbols) of TPP þ absorption kinetics of (1) treated yeast cells with 150 ms duration 4.8 kV/cm PEF (adjusted R2 ¼ 0.863) and (2) untreated yeast cells (adjusted R2 ¼ 0.989). The solid curves were obtained using Equation (2).
duration and electric field strengths are presented in Figure 3 (symbols). The measurements of residual TPPþ concentration in the buffer were performed after 3 min of incubation. Therefore, the dependences of TPPþ absorption amount on electric field strength are represented in Figure 3 above 9% level (dashed line),
Fig. 3. The effect of strength of PEF onTPP þ accumulation in the cells.The experimental data (symbols) show how theTPP þ accumulation depends on the electric field strength after t ¼ 3 min since the PEF treatment at different square shaped electric pulse duration. The solid curves were obtained using Equation (4). Adjusted R2 wasobtainedintherange of 0.899^0.973.The dashed line represents the amount of TPP þ which was absorbed by yeastupto 3 minafter treatment by PEF.Errormarginsare10%. Bioelectromagnetics
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duration, the TPPþ absorption by the yeast is increased and tends to saturation when the applied PEF exceeds 9 kV/cm. It is evident that for different pulse durations, these dependences become observable at a certain critical electric field strength called threshold electric field (Eth p). Thus, for the acceleration of the rate of TPPþ accumulation by PEF, the electric field higher than Eth p has to be reached. The Eth p is a function of the electric pulse duration (tp) and decreases with an increase of tp (see Fig. 4). The Eth p at which the TPPþ absorption starts to increase is inversely proportional to the square root of the electric pulse duration tp. The inset confirms such relation.
Fig. 4. The dependence of the critical electrical field strength (Eth p) on the pulse duration. The inset presents the same Eth p values as a function of (tp) 0.5.
which corresponds to the TPPþ absorption up to 3 min. As can be seen in Figure 3, the TPPþ absorption efficiency definitely depends on the strength and pulse duration of the applied electric field. By increasing the PEF strength at constant pulse
Plasma Membrane Permeability and Viability of PEF-Exposed Yeast Cells Figure 5 presents the evaluation of the metabolic activity of the yeast cells after PEF exposure. The unaffected yeast cells converted the substrate to an orange colored formazan-based compound to 1.3 0.2 OD (Fig. 5, left arrow, squares), that is, proportional to the concentration of the dye. As it was already mentioned, the concentration of the dye was determined by measuring the optical absorbance. When the PEF strength using a single 150 ms duration pulse was increased and exceeded 5.82 kV/cm the cell proliferation decreased dramatically. This point is the viability threshold of the S. cerevisiae SEY6210 yeast. When
Fig. 5. The evaluation of the permeabilization and the metabolic activity of the yeast cells after PEF exposure. The fraction of electropermeabilized yeast cells was determined by the calcein blue AM release assay and the determination of the yeast viability using the cell proliferation assay where each point is the mean of the three experiments. All experiments were performed using single 150 ms duration pulses. The vertical arrow represents the yeast viability threshold field. Bioelectromagnetics
PEF-Induced Yeast Cell Wall Permeabilization
the strength of the electric pulse was increased to 10 kV/cm, the changes of the optical absorbance decreased to about 0.3 0.02 OD, which indicated a decrease of the viable cell density due to cell death or slowing of their metabolism. The dye release assay was performed using fluorescent microscopy. The PEF influences the calcein blue effluxes through the hydrophilic pores in the cell membrane. The count of the yeast cells which were permeable to calcein was conducted after treating the cell suspension with 150 ms pulses having electric field strength of 0, 2, 4, 5.82, 7.7, 8.7, and 9.7 kV/cm, respectively. The cell membrane permeability increased when the strength of the electric field exceeded 3 kV/cm and was tending to saturation. About 80% of the yeast cells were identified as permeable to the calcein blue dye when the electric field strength was more than 8 kV/cm (Fig. 5, right arrow, circles). Additionally, the fluorescent images of the yeast cell suspension were performed (see next section, inset of Fig. 6). Modeling of TPPþ Absorption The experimental investigation of TPPþ absorption by yeast proved that the rate of TPPþ accumulation was accelerated by PEF, because the TPPþ concentration in the suspension decreased up to several times compared with that in the initial suspension (Fig. 3). Measurements of the residual TPPþ concentration in the buffer solution with the cells after PEF exposure and incubation for 3 min were performed. In this case, the kinetics of the TPPþ
Fig. 6. The dependence of parameter B on the pulse duration. Adjusted R2 ¼ 0.962. The inset shows the calcein blue AM stained yeast cell aggregates, which were dispersed after PEF exposure.
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absorption process can be described using the second order rate equation, which takes into account that the concentration of TPPþ molecules dispersed in the buffer solution changes during their absorption by the yeast, as defined by its “finite source” condition. This condition corresponds to the measurement method that detects changes of TPPþ concentration in buffer solution. It is known that the TPPþ accumulation process by the yeasts is very slow and the equilibrium is reached in 120 min [Ballarin-Denti et al., 1994]. Therefore, the influence of expulsion from the yeast can be omitted from the accumulation process because TPPþ recovery to the buffer free of TPPþ is much slower. For this reason TPPþ accumulation process can be described by the following equation [Neumann et al., 1996]: dN ¼ k a ðN m N ÞðN s N Þ dt
ð1Þ
where N is the number of the TPPþ molecules per volume unit (concentration) absorbed by the yeasts; (Nm N) is a concentration of free absorption sites in the yeasts; (Ns N) is a concentration of available TPPþ molecules within the buffer solution; ka is a coefficient, which quantifies the rate of the absorption process. The solution of Equation (1) in the case when Nm/Ns
Electric Field-Induced Effects on Yeast Cell Wall Permeabilization Arunas Stirke,1,2* Aurelijus Zimkus,1,3 Almira Ramanaviciene,2 Saulius Balevicius,1 Nerija Zurauskiene,1 Gintautas Saulis,1 Larisa Chaustova,3 Voitech Stankevic,1 and Arunas Ramanavicius1,2 1 Center for Physical Sciences and Technology,Vilnius, Lithuania Center of Nanotechnology and Materials ScienceNanoTechnas, Faculty of Chemistry, Vilnius University,Vilnius, Lithuania 3 Department of Biochemistry and Biophysics,Vilnius University,Vilnius, Lithuania
2
The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPPþ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 ms and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPPþ absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPPþ by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPPþ absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPPþ, is due to the poration of the plasma membrane. The applicability of the TPPþ absorption measurements for the analysis of yeast cells electroporation process is also discussed. Bioelectromagnetics 35:136–144, 2014. © 2013 Wiley Periodicals, Inc. Key words: electroporation; tetraphenylphosphonium; pulsed electric field; yeast
INTRODUCTION For several decades, the budding yeasts (Saccharomyces cerevisiae) have been considered prototypical of eukaryotic cells, ideally suited for use in studies of many of the basic phenomena of eukaryotic life and some of their fundamental properties [Neumann et al., 1996; Breitenbach et al., 2012]. Yeasts are used not only to produce transgenic proteins, or for fermentation in foods and beverages, but also can be used as microbial fuels cells for generating green electricity [Ramanavicius and Ramanaviciene, 2009; Liu et al., 2011]. The S. cerevisiae yeast cells are surrounded by cell walls, which provide them with protection from osmotic stress and are also important for their defense against toxic compounds, for self-recognition and for flocculation [Aouida et al., 2003; Veelders et al., 2010; Levin, 2011]. Yeast cell walls are mainly composed of fibrous polysaccharide b-1,3-glucan and mannoproteins (proteins highly N-and O-glycosylated with mannose residues), which are located on the external 2013 Wiley Periodicals, Inc.
layers of the walls. Such mannosyl side chains are phosphorylated and provide an anionic surface charge to the yeast cell wall surfaces at physiological pH values [Jigami and Odani, 1999; Van Holle et al., 2012]. The cell wall hydrophobicity and charge are the major determinants of yeast adhesion on abiotic surfaces. Both play major roles in medical and bioprocessing industries as they form biofilms on human tissue and biofouling surfaces when used in the Grant sponsor: Lithuania Agency for Science, Innovation and Technology; grant number: 31V-33. *Correspondence to: Arunas Stirke, Center for Physical Sciences and Technology, A. Gostauto 11, LT-01108, Vilnius, Lithuania. E-mail: [email protected] Received for review 18 February 2013; Accepted 19 September 2013 DOI: 10.1002/bem.21824 Published online 6 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
PEF-Induced Yeast Cell Wall Permeabilization
food or beverage industries [Rhymes and Smart, 2001; White and Walker, 2011]. Several methods have been used to change the permeability of the yeast cells. These include treatment of the cells chemically by adding reducing agents such as dithiothreitol (DTT) or antibiotics [Brajtburg et al., 1990; Klis et al., 2007]. Their permeability can also be changed by increasing the external mechanical pressure on the cells [Smith et al., 2000], or by treating them with pulsed electric fields (PEFs; electropermeabilization or electroporation), which facilitates the leakage from the cells of intracellular compounds, such as proteins, ions, and nucleic acids [Liu et al., 2011]. In order to detect changes of the permeability of the yeast cells, fluorescent probes are used. Various charged molecule adsorption measurements are taken to determine the changes induced in the yeast cell wall surface charge by the PEF [Tomov and Tsoneva, 1989; Neumann et al., 1996]. Bleomycin has also been used as a probe in yeast viability studies affected by PEF. This enables analysis of the contributions of the cell wall and the cell membrane in limiting the uptake of a drug [Aouida et al., 2003]. Bleomycin is an antitumor drug, a hydrophilic molecule that is unable to cross the cell membrane by free diffusion, but this drug also has a positively charged tail [Chen and Stubbe, 2005]. For this reason, the molecule can be retained on the surface of the yeast cell. Other researchers have used indirect methods (electric conductivity of the medium and zpotential of yeasts cells) to analyze permeabilization and have proposed some hypothetical assumptions on the mechanism of the surface charge changes of yeast cells affected by PEF [El Zakhem et al., 2006a]. However, the quantitative measurement of the cell permeabilization using these methods is difficult. Moreover, only few methods can be used for the indication of selective cell wall permeabilization without causing lethal influences to the cells. One of the possible ways of increasing this selectivity is the application of lipophilic cations (LCs) as a probe for the indication of the permeability of the yeast cell walls. Tetraphenylphosphonium salts (e.g., tetraphenylphosphonium bromide [TPPBr]) are frequently used in the measuring the membrane potential (Dc) of prokaryotic and eukaryotic cells. The use of a potentiometric ion-selective electrode is also a convenient method for the quantitative evaluation of the permeability of the yeast cell wall and membrane [Zimkus and Chaustova, 2004]. The purpose of this study was to investigate the permeability of viable yeast cells after exposure to PEF using the indirect TPPþ selective probe-based method.
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MATERIALS AND METHODS Yeast Strain and Cultivation S. cerevisiae SEY6210 (MATa, leu2-3, leu2-112, ura3-52, his3-D200, trp1-D901, lys2-801, suc2-D9, GAL) yeast cells (donated by Prof. H. Bussey, McGill University, Quebec, Canada) were grown in a 250 ml of complete medium of yeast extract-peptone-dextrose (YPD; 1% yeast extract, 2% peptone, 2% glucose, Sigma–Aldrich, St. Louis, MO) at 30 8C in a reciprocal shaker operating at 150 rpm. Cell density was measured by optical density (OD) using an optical absorption spectrophotometer (Biophotometer; Eppendorf, Hamburg, Germany) operating at a wavelength of 600 nm. In order to collect the cell more sensitive to the physical treatments [Anton-Leberre et al., 2010] and to ensure the repeatability of results, the yeast cells were centrifuged when the OD achieved 0.8–1.0 for light path 1 cm. At the early exponential growth phase yeast cells were washed with distilled water and then resuspended in 1 ml of cold (4 8C) electroporation buffer (EPB) containing 1 mol/dm3 sorbitol 20 mmol/ dm3 Tris–HCl buffer, pH 7.4 (Sigma–Aldrich) keeping the final concentration of the yeasts at (4–6) 109 colony forming unit (CFU)/ml [Zimkus et al., 2006]. Electroporation Setup The experimental setup for the electroporation of the yeast is presented in Figure 1. It consisted of a high voltage DC power supply (HV source) ALE 500A (TDK-Lambda Americas, Neptune, NJ), a low voltage, microsecond square-wave pulse generator TG 5011 (Thurlby Thandar Instruments, Cambridgeshire, UK), a high voltage electrical pulse forming circuit (Fig. 1, shaded area), a real time oscilloscope DPO4034 (Tektronix, Beaverton, OR) and a commercial cuvette (Lonza, Cologne, Germany). The variable HV source was used to charge a 120 mF capacitor C up to a voltage from several volts to 1 kV. The charging of capacitors was performed through the charging resistor RCh ¼ 100 kV. The pulse generator creates square-wave pulses that were used for control of an isolated bipolar transistor (IGBT) 1MBH25D120 (Fuji Electric, Tokyo, Japan). During the experiments, pulse duration was varied from 0.1 ms to 1 ms. The circuit operated as follows: after charging the capacitor bank C, the square-wave pulse generator creates a required duration trigger pulse, which triggers the IGBT through a HCPL3120 gate driver (Avago Technologies, San Jose, CA). The IGBT was capable of withstanding collector-emitter voltages up to 1200 V and current pulses of lasting 1 ms with maximal amplitudes up to 114 A. The input circuit of the gate driver was optically isolated. A low Bioelectromagnetics
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Fig.1. The experimental setup used for yeast cell electroporation. The shaded area is a high voltage electrical pulse forming circuit which consists of the isolated gate bipolar transistor (IGBT), gate driver of this transistor, capacitor (C), ballast resistor (RB), capacitors charging resistor (RCh), and shunt resistor (RSh).
inductance ballast resistor RB ¼ 20 V was used to protect IGBT transistor from current overshoot. When this IGBT was switched on, the capacitor discharged a cuvette through the load with a buffer containing the yeast and ballast resistor. A real time oscilloscope connected in parallel to the shunt resistor RSh ¼ 2 V was used for the measurement and calculation of the pulse parameters: duration and waveform of current through the cuvette and electric field strength. The typical waveform of the measured pulse (VOsc) is presented in the inset of Figure 1. The rise time was 0.1 ms and the fall time was approximately 0.15 ms. It has to be noted that the shape of this waveform represents current through the cuvette (voltage measured across shunt resistor). Good square shape indicates that conductivity of the buffer with the yeast does not change during the pulse (no heating, electrolysis or other processes are observed during the pulse). The strength of the PEF Ep across the cuvette was calculated according to the equation Ep ¼ Vp/d; where d is the distance between the electrodes; Vp is the voltage drop across the cuvette: V p ¼ V S ðV Osc =RSh ÞðRSh þ RB Þ, where the VS is the power source voltage. A commercial cuvette 120 ml of total volume with plate-shape electrodes of 2 cm2 and gap between electrodes of 1 mm was used. Bioelectromagnetics
TPPþ Accumulation Analysis All electroporation procedures were carried out in abovementioned commercial cuvette filled with 120 ml of yeast suspension prepared in EPB. TPPBr to a final concentration of 5 106 mol/dm3 was added before the electroporation. After electroporation and incubation for an additional 3 min, the test samples were precipitated and TPPþ absorption measurements were performed in separated tubes by mixing 50 ml of resultant supernatant with 200 ml of electrode calibration solution (TPPþ concentration 2 106 mol/dm3). After that initial TPPþ concentration Ns was [TPPþ]ini6 mol/dm3. Such solution was used for the tial ¼ 2.6 10 measurement of the residual TPPþ ion concentration in supernatant ([TPPþ]supernatant). The quantity (N) of the TPPþ absorbed by the yeast cells was calculated as follows: N ¼ [TPPþ]initial [TPPþ]supernatant, that is, N ¼ Ns Nsupernatant. The control sample was prepared and measured at 0 kV/cm as well as test samples, which were treated using single square shape electric field pulses of 5, 10, 50, 100, and 150 ms duration and different electric field strengths up to 10 kV/cm. A combined tetraphenylphosphonium selective electrode was used for this purpose. This electrode has been described previously [Zimkus and Chaustova, 2004]. The electrode potential drift was estimated using a WTW 526 ionometer (WTW, Weilheim, Germany) connected to a PC. In order to investigate the TPPþ accumulation level, these measurements were conducted at different time intervals after electroporation and incubation of the EPB with TPPþ at room temperature (20 8C). Evaluation of the Proliferation of Yeast Cells After the Action of PEF The determination of the yeast cell viability was conducted before and after the electroporation procedure. The yeast proliferation assay was conducted using a microbial cell proliferation kit based on absorption changes of 2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8; Dojindo, Kumamoto, Japan) after an action of viable yeast cells. Two different mediums were used: an EPB buffer and a synthetic complete (SC) medium (colorless yeast nitrogen base medium; Sigma–Aldrich) with 2% of glucose (Merck, Darmstadt, Germany). After incubation of the yeast cells for 4 h at 30 8C, measurements of the absorption at a 460 nm wavelength were conducted with a Tecan Sunrise microplate reader (Tecan Group, Männedorf, Switzerland). The obtained results of the three experiments are presented as means with standard deviation.
PEF-Induced Yeast Cell Wall Permeabilization
The Dye Release Assay To determine the effectiveness of the plasma membrane electroporation, a calcein blue release assay was performed. The yeast cells were concentrated to 1.5 108 CFU/ml in 100 ml of the EPB buffer and then 400 mmol/dm3 of acetoxymethyl (AM) C1429 esterified calcein blue (Life Technologies, Eugene, OR), a cell permeable derivate of calcein blue, was added. After 30 min of incubation at room temperature (20 8C) and avoiding any exposure to light, the cells were observed using a BX51TF fluorescence microscope (Olympus, Tokyo, Japan) equipped with an appropriate filter cube. Images of the yeast cells were recorded using an Evolution QEi Monochrome camera (Media Cybernetics, Silver Spring, MD) and were processed using a Pro Express v.6 (Media Cybernetics) program. Measuring the percentages of fluorescent and non-fluorescent yeast cells in the cuvette using the dye release test determined the electroporation efficiency of the PEF. This test is based on the fact that the electroporated cells are practically permeable to such dyes as calcein. At least 100 yeast cells were counted in each experiment. The experiments were repeated three times. RESULTS The Accumulation of TPPþ by the Yeast Cells After Exposure to PEF The quantity of the TPPþ absorbed by the yeast cells was expressed as accumulation level and was given by the ratio (N/Nm) 100%. Here Nm is the maximal concentration of the molecules that can be accumulated inside the yeasts (steady-state accumulation level). Initial concentration of TPPþ (Ns) was always kept higher than Nm. It was determined that the concentration of the TPPþ accumulated in the yeasts cells depends on the incubation time of the cells with the LCs. For the various yeast strains, a steady-state accumulation level Nm is reached after a few hours of treatment in the buffer solutions [Zimkus and Chaustova, 2003]. The rate of the LC absorption of the untreated yeast cells is lower in comparison to the ones treated by the PEF. A TPPþ accumulation level of 90% was reached after 100 min for the untreated cells and after 40 min for the yeast cells treated with a 4.8-kV/cm electric field strength, 150 ms duration pulse (see Fig. 2, symbols). It has to be noted that the steady-state accumulation level is not changed by the PEF action on the yeast cells. The results of the TPPþ accumulation by the yeast cells that were obtained after treating them with a single square shape pulses having different
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Fig. 2. The experimental data (symbols) of TPP þ absorption kinetics of (1) treated yeast cells with 150 ms duration 4.8 kV/cm PEF (adjusted R2 ¼ 0.863) and (2) untreated yeast cells (adjusted R2 ¼ 0.989). The solid curves were obtained using Equation (2).
duration and electric field strengths are presented in Figure 3 (symbols). The measurements of residual TPPþ concentration in the buffer were performed after 3 min of incubation. Therefore, the dependences of TPPþ absorption amount on electric field strength are represented in Figure 3 above 9% level (dashed line),
Fig. 3. The effect of strength of PEF onTPP þ accumulation in the cells.The experimental data (symbols) show how theTPP þ accumulation depends on the electric field strength after t ¼ 3 min since the PEF treatment at different square shaped electric pulse duration. The solid curves were obtained using Equation (4). Adjusted R2 wasobtainedintherange of 0.899^0.973.The dashed line represents the amount of TPP þ which was absorbed by yeastupto 3 minafter treatment by PEF.Errormarginsare10%. Bioelectromagnetics
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duration, the TPPþ absorption by the yeast is increased and tends to saturation when the applied PEF exceeds 9 kV/cm. It is evident that for different pulse durations, these dependences become observable at a certain critical electric field strength called threshold electric field (Eth p). Thus, for the acceleration of the rate of TPPþ accumulation by PEF, the electric field higher than Eth p has to be reached. The Eth p is a function of the electric pulse duration (tp) and decreases with an increase of tp (see Fig. 4). The Eth p at which the TPPþ absorption starts to increase is inversely proportional to the square root of the electric pulse duration tp. The inset confirms such relation.
Fig. 4. The dependence of the critical electrical field strength (Eth p) on the pulse duration. The inset presents the same Eth p values as a function of (tp) 0.5.
which corresponds to the TPPþ absorption up to 3 min. As can be seen in Figure 3, the TPPþ absorption efficiency definitely depends on the strength and pulse duration of the applied electric field. By increasing the PEF strength at constant pulse
Plasma Membrane Permeability and Viability of PEF-Exposed Yeast Cells Figure 5 presents the evaluation of the metabolic activity of the yeast cells after PEF exposure. The unaffected yeast cells converted the substrate to an orange colored formazan-based compound to 1.3 0.2 OD (Fig. 5, left arrow, squares), that is, proportional to the concentration of the dye. As it was already mentioned, the concentration of the dye was determined by measuring the optical absorbance. When the PEF strength using a single 150 ms duration pulse was increased and exceeded 5.82 kV/cm the cell proliferation decreased dramatically. This point is the viability threshold of the S. cerevisiae SEY6210 yeast. When
Fig. 5. The evaluation of the permeabilization and the metabolic activity of the yeast cells after PEF exposure. The fraction of electropermeabilized yeast cells was determined by the calcein blue AM release assay and the determination of the yeast viability using the cell proliferation assay where each point is the mean of the three experiments. All experiments were performed using single 150 ms duration pulses. The vertical arrow represents the yeast viability threshold field. Bioelectromagnetics
PEF-Induced Yeast Cell Wall Permeabilization
the strength of the electric pulse was increased to 10 kV/cm, the changes of the optical absorbance decreased to about 0.3 0.02 OD, which indicated a decrease of the viable cell density due to cell death or slowing of their metabolism. The dye release assay was performed using fluorescent microscopy. The PEF influences the calcein blue effluxes through the hydrophilic pores in the cell membrane. The count of the yeast cells which were permeable to calcein was conducted after treating the cell suspension with 150 ms pulses having electric field strength of 0, 2, 4, 5.82, 7.7, 8.7, and 9.7 kV/cm, respectively. The cell membrane permeability increased when the strength of the electric field exceeded 3 kV/cm and was tending to saturation. About 80% of the yeast cells were identified as permeable to the calcein blue dye when the electric field strength was more than 8 kV/cm (Fig. 5, right arrow, circles). Additionally, the fluorescent images of the yeast cell suspension were performed (see next section, inset of Fig. 6). Modeling of TPPþ Absorption The experimental investigation of TPPþ absorption by yeast proved that the rate of TPPþ accumulation was accelerated by PEF, because the TPPþ concentration in the suspension decreased up to several times compared with that in the initial suspension (Fig. 3). Measurements of the residual TPPþ concentration in the buffer solution with the cells after PEF exposure and incubation for 3 min were performed. In this case, the kinetics of the TPPþ
Fig. 6. The dependence of parameter B on the pulse duration. Adjusted R2 ¼ 0.962. The inset shows the calcein blue AM stained yeast cell aggregates, which were dispersed after PEF exposure.
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absorption process can be described using the second order rate equation, which takes into account that the concentration of TPPþ molecules dispersed in the buffer solution changes during their absorption by the yeast, as defined by its “finite source” condition. This condition corresponds to the measurement method that detects changes of TPPþ concentration in buffer solution. It is known that the TPPþ accumulation process by the yeasts is very slow and the equilibrium is reached in 120 min [Ballarin-Denti et al., 1994]. Therefore, the influence of expulsion from the yeast can be omitted from the accumulation process because TPPþ recovery to the buffer free of TPPþ is much slower. For this reason TPPþ accumulation process can be described by the following equation [Neumann et al., 1996]: dN ¼ k a ðN m N ÞðN s N Þ dt
ð1Þ
where N is the number of the TPPþ molecules per volume unit (concentration) absorbed by the yeasts; (Nm N) is a concentration of free absorption sites in the yeasts; (Ns N) is a concentration of available TPPþ molecules within the buffer solution; ka is a coefficient, which quantifies the rate of the absorption process. The solution of Equation (1) in the case when Nm/Ns
