Role of glutathione in ethanol stress tolerance in yeast Pachysolen tannophilus

Biochemical and Biophysical Research Communications 397 (2010) 307–310

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Role of glutathione in ethanol stress tolerance in yeast Pachysolen tannophilus Rajesh Kumar Saharan *, Smita Kanwal, Sukesh Chander Sharma Department of Biochemistry, Panjab University, Chandigarh, India

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Article history: Received 4 May 2010 Available online 26 May 2010 Keywords: Oxidative stress tolerance Pachysolen tannophilus Glutathione

a b s t r a c t The effect of glutathione enrichment and depletion on the survival of Pachysolen tannophilus after ethanol stress was investigated. In this work, we verified that both control and glutathione deficient yeast cells were much more oxidized after ethanol stress. Depletion of cellular glutathione enhanced the sensitivity to ethanol and suppressed the adaptation. Incubation of the cell with amino acids constituting glutathione (GIu, Cys, Gly) increased the intracellular glutathione content, and subsequently the cell acquired resistance against ethanol. The level of reactive oxygen species, protein carbonyl, and lipid peroxidation in glutathione enriched groups were also studied. These results strongly suggest that intracellular glutathione plays an important role in the adaptive response in P. tannophilus to ethanol induced oxidative stress. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Studies of the cellular responses to metabolic stresses are not only a subject for microbial physiologists and ecologists wishing to understand basic cellular mechanisms, but also a crucial subject for microbial technologists who have to maintain metabolic viability and vitality of microbial strains used in industrial fermentations. This is, in fact, a matter of survival for the continued success of traditional food and beverages and also for presentday biopharmaceuticals biotechnologies. The biotechnology, food, and pharmaceutical industries employ many Saccharomyces but also non-Saccharomyces yeasts [1]. This already long and profitable association has created the necessity of a better understanding of metabolic peculiarities of non-conventional yeasts (NCY) during various kind of stress during the process of fermentation. These stresses may result in the impairment of the strains’ growth and metabolism and seriously affect industrial companies. The yeast Pachysolen tannophilus, for example, is known to elicit different adaptive stress response mechanisms in an effort to survive and thrive in hostile conditions [2]. Common stresses include pH and temperature shocks; oxidative stress; osmotic shock; and fermentation products toxicity, mainly ethanol [3,4]. Previous studies have shown that oxidative stress may be responsible for ethanol caused cell damage because of the excess generated ROS [5]. Cells

possess an inherent protective mechanism to control the level of ROS, like antioxidative enzymes SOD, CAT, glutathione peroxidase, and intracellular non-enzymic molecules like glutathione [6,7]. Glutathione are cysteine-rich proteins that provide protection against oxidative stress [8,9]. Induction of glutathione synthesis by oxidative and heat stress is well studied [10,11]. However, information about the role of glutathione on the ethanol stress response in yeast is largely lacking. Much previous work about ethanol stress responses of yeast has focused on stress-inducible proteins and membrane lipid unsaturation [12]. In this study, we examined the function of intracellular glutathione on ethanol stress response in yeast. GSH is implicated in a detoxification mechanism of xenobiotics [13], oxidative stress [14], and heavy metal stress [15,16]. While it seems obvious that as GSH is implicated in numerous stress response mechanisms, the tripeptide may also play a role in the maintenance of basic functions during ethanol stress. Diethyl maleate (DEM) is an inhibitor of glutathione synthesis has been used in this study to deplete GSH and enrichment of the glutathione was done by the incorporation of amino acid (GIu, Cys, Gly) in the media. We investigated whether the depletion of GSH would alter the ethanol tolerance to yeast cells and compare it with the glutathione enriched cells. 2. Materials and methods

Abbreviations: DTNB, 5,50 -dithiobis-(2-nitrobenzoic acid); PBS, phosphatebuffered saline; GSH, reduced glutathione; GSSG, total glutathione; GIu, L-glutamic acid; Cys, L-cysteine; Gly, glycine; CAT, catalase; SOD, superoxide dismutase; ROS, reactive oxygen species; DEM, diethyl maleate. * Corresponding author. Address: Department of Biochemistry, Panjab University, Chandigarh 160014, India. Fax: +91 172 2541022. E-mail address: [email protected] (R.K. Saharan). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.107

2.1. Organism and culture condition Yeast strain P. tannophilus, Y1038 (procured from IIT, Delhi) was maintained at 4 °C on YPDA (2% dextrose, 1% yeast extract, 2% peptone, and 2% agar). The cells were grown in YPD media (pH 5.5) in

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Erlenmeyer flasks with liquid-to-air volume ratio of 1:5 at 200 rpm and 30 °C. 2.2. Assay of cell resistance to ethanol To observe the sensitivity of yeast to ethanol, the ethanol was added in YPD media that contains cells of exponential phase (approximately 1–2  107 cells ml1) to make final concentration 10% (v/v), in Erlenmeyer flasks. After incubation at 30 °C and 100 rpm for various time intervals, fixed aliquots were withdrawn with autopipette. 2.3. Assay of glutathione enrichment and depletion For enrichment of glutathione content, cells were incubated in a mixture containing 0.5 M glucose, 0.01 M MgC12, 0.02 M L-glutamic acid, 0.02 M L-cysteine, 0.02 M glycine, and 0.1 M potassium phosphate buffer (pH 7.4) at 28 °C for 1 h with shaking [17]. For depletion of glutathione content, cells were incubated with the 5 mM DEM at 28 °C for 1 h with shaking [18]. After the incubation, cells were treated with the ethanol. 2.4. Glutathione determination For determination of glutathione, samples were suspended in the 10% TCA and vortexed for 10 min to extract glutathione. After centrifugation at 10,000g for 30 min reduced glutathione (GSH) was estimated in supernatant according to the method of Beutler et al. [19]. This method is based on reaction of –SH groups of reduced glutathione with 5,50 -dithio-bis-2-nitrobenzoic acid. The total glutathione content of was estimated by the method of Habeeb [20]. Levels of oxidized glutathione (GSSG) were calculated from the difference between the values of total glutathione and reduced glutathione (GSH) and the results were expressed as lg/mg protein. 2.5. Evaluation of lipid peroxidation Lipid peroxidation was quantified by thiobarbituric acid (TBA)reactive substances (TBARS). TBARS was determined according to the method described by Buege and Aust [21]. At certain intervals 0.5-ml samples of cell suspension were removed and added to 1 ml of TBA reagent (15% wt./vol. TCA and 0.375% wt./vol. TBA in 0.25 M HCl). Addition of the reagent terminated lipid peroxidation and initiated the assay. Samples were heated for 15 min in a boiling water bath and, after cooling, were centrifuged at 10,000g for 5 min in order to remove cell debris. Absorbance of the supernatant at 535 nm were measured by using a Shimadzu UV-1240 spectrophotometer, against a reference solution comprising 1 ml of TBA reagent with the sample replaced by an equal volume (0.5 ml) of distilled deionized water. The concentrations of TBARS in samples were calculated by using molar extinction coefficient of MDA–thiobarbituric chromophore 1.56  105 M1 cm1.

aliphatic hydrazones of 22,000 M1 cm1 and expressed as nmol carbonyl/mg of protein. 2.7. Measurement of intracellular oxidation level Intracellular ROS was detected by the oxidant-sensitive probe 20 ,70 -dichlorofluorescein diacetate (DCFH-DA, Sigma) [23]. According to this procedure 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) was added from a fresh 5 mM stock (prepared in ethanol) to a final concentration of 10 mM in 1 ml of yeast cell culture (107 cells) then incubated at 28 °C for 20 min. Finally, cells were cooled on ice, harvested by centrifugation and washed twice with distilled water. The pellet was resuspended in 500 lL of water and 1.5 g of glass beads were added. Cells were lysed by three cycles of 1 min agitation on a vortex mixer followed by 1 min on ice. The supernatant was obtained after centrifugation at 25,000g for 5 min. and after appropriate dilution with water the fluorescence was measured using a Shimadzu Spectrofluorophotometer (RF-5301PC) with excitation at 502 nm and emission at 521 nm. 2.8. Viability measurement Viability of the cells was determined by colony counting after spreading appropriate dilutions in duplicate on YPD-agar, following incubation at 30 °C for 3 days. The percent viability was calculated with respect to cells grown without ethanol. 2.9. Protein estimation The pellet obtained after trehalose and glutathione extraction was solubilized by boiling for 5 min with 2.0 ml of 0.1 M NaOH. The clear supernatant was used for protein estimation by Lowry method [24] using bovine serum albumin as standard. 3. Results and discussion 3.1. Ethanol stress induces an oxidative stress, which seems to affect the viability of yeast In this work, oxidative stress produced by ethanol stress was monitored by measuring changes in fluorescence resulting from

2.6. Quantification of protein bound carbonyls For assessment of carbonyls the reaction with dinitrophenylhydrazine was employed [22]. For each determination, samples containing 2–10 mg ml1 of protein were treated with 4 ml of 10 mM dinitrophenylhydrazine in 2.5 M HCl for 1 h at room temperature. One tube, used as the blank, was incubated only with 2.5 M HCl. The reaction was stopped by addition of 5 ml of 20% TCA. The pellets were washed twice with 3 ml of absolute ethanol/ethyl acetate (1:1) solution. The protein pellets were finally dissolved in 6 M guanidine hydrochloride and the absorption at 375 nm (dinitrophenylhydrazine minus sample blank) was determined. Carbonyl content was calculated using the molar absorption coefficient of

Fig. 1. Ethanol response detected by 20 ,70 -dichlorodihydrofluorescein diacetate assay. Fluorescence of the cell extracts from control cells (black), glutathione depleted cells (white), and glutathione enriched (gray) cells. The bars represent the fluorescence intensity in arbitrary units (AU). Each experiment was done at least in triplicate.

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oxidation of an intracellular probe. The probe 20 ,70 -dichlorofluorescein diacetate is a molecule that can permeate cell membranes by passive diffusion. Once inside the cell, it becomes susceptible to attack by free radical species, producing a more fluorescent compound [25]. The intensity of fluorescence can be measured and it is the basis of a commonly used assay for oxidative stress. According to the results (Fig. 1), control and glutathione deficient cells showed a meaningful increase in oxidation but there is less oxidation observed in the glutathione enriched cells after 1 and 2 h ethanol stress, confirming that oxidative stress plays a major role in the toxic effects of ethanol stress. Glutathione is dispensable for growth under oxidative stress conditions [26]. A crucial strategy for survival in ethanol stress condition is the control of membrane dynamics [27–29]. Under stress, cell membranes become fluidized and perturbed, as well as being more susceptible to ROS attack [30]. ROS often cause an extensive peroxidation and de-esterification of membrane lipids at intermediate ranges of water loss. In yeast during various stresses glutathione involved in peroxide reduction and removal of many lipophilic xenobiotics from the cytosol [31], it seems that GSH is important in protecting cell membranes under anhydrous conditions [32]. There are several ways to detect lipid peroxidation processes; we used the method of TBARS (thiobarbituric acid reactive species), which detects malondialdehyde (MDA). Ethanol stress produced increased levels of lipid peroxidation in both control and glutathione depleted cells (Table 1). However, corroborating the idea that GSH might protect membrane against oxidation, the levels of lipid peroxidation in the glutathione enriched cells were lesser than in the control cells and glutathione depleted cells. As expected, after ethanol stress, there was a significant decline of the intracellular GSH content in the control cells and glutathione depleted cells as well as in the glutathione enriched cells after ethanol stress (Table 2), confirming once again the key role played by glutathione in maintaining the redox homeostasis of ethanol stressed cells. To confirm the role of glutathione in the oxidative stress caused by the ethanol, protein carbonyl formation was studied. The increased level of protein carbonyl was observed in the control and glutathione depleted cells in comparison with the glutathione enriched cells (Fig. 2). Proteins carbonyls are toxic for cells and they

Fig. 2. Ethanol response detected by protein carbonyl formation assay. Protein carbonyl of the cell extracts from control cells (black), glutathione depleted cells (white), and glutathione enriched (gray) cells. Each experiment was done at least in triplicate.

should be recognized and degraded by cellular proteolytic processes. Failure to control on early protein oxidation may allow for oxidized proteins to become more severely oxidized. These indigestible protein aggregates are toxic and have been linked to the initiation and progression of cell death [33]. The mitochondria proteins carbonyl formation further deteriorate the situation by

Table 1 Effect of ethanol stress on lipid peroxidation of Pachysolen tannophilus with altered glutathione level and its comparison with the control group treated with the ethanol. Time

0 min

60 min

Lipid peroxidation (TBAR species nM/mg protein) Control cells + 10% ethanol 0.230 ± 0.006 0.276 ± 0.005 GSH () cells + 10% ethanol 0.453 ± 0.012 0.501 ± 0.005 GSH (+) cells + 10% ethanol 0.236 ± 0.0043 0.265 ± 0.004

120 min 0.331 ± 0.009 0.503 ± 0.005 0.284 ± 0.004

The results represent means ± SD of three independent experiments. Abbreviations: , represents depleted glutathione level; +, represents increased glutathione level.

Fig. 3. Percentage viability after ethanol stress of control cells (black), glutathione depleted cells (white), and glutathione enriched cells (gray). The value is mean of three independent experiments.

Table 2 Effect of ethanol stress on glutathione level of Pachysolen tannophilus with altered glutathione level and its comparison with the control group treated with the ethanol. Reduced GSH (lg/mg protein)

Oxidized GSH (lg/mg protein)

Reduced/oxidized GSH

Time (min)

0

60

120

0

60

120

0

60

120

Control cells + 10% ethanol GSH () cells + 10% ethanol GSH (+) cells + 10% ethanol

17.06 ± 0.40 4.02 ± 0.31 38.13 ± 0.70

11.31 ± 0.59 2.38 ± 0.41 10.74 ± 0.56

7.93 ± 0.30 1.23 ± 0.19 18.86 ± 0.29

16.93 ± 0.44 5.16 ± 0.45 5.97 ± 0.64

16.07 ± 0.99 2.77 ± 0.54 9.76 ± 0.62

18.28 ± 0.57 2.06 ± 0.35 15.07 ± 0.79

1.004 0.779 6.384

0.704 0.860 1.101

0.434 0.598 1.252

The results represent means ± SD of three independent experiments. Abbreviations: , represents depleted glutathione level; +, represents increased glutathione level.

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decreased ATP synthesis and enhanced formation of ROS. Glutathione plays a pivotal role in many metabolic activities [34], so reduction in the level of glutathione may be responsible for decreased cell viability due to inactivation of many essential proteins. The lessen protein carbonyl formation and increased viability in glutathione enriched cells during ethanol stress proves the role of glutathione in the ethanol tolerance. As seen in Fig. 3 cells lacking glutathione, showed decreased viability than the control and glutathione enriched cells. We conclude, therefore, that glutathione plays a remarkable role in protecting cell membranes and in maintaining redox homeostasis under ethanol stress, favoring tolerance to the stress state. However, the study of the ability to survive in ethanol stress conditions indicates that other aspects are also important for conferring tolerance and long-term stability. It has often been found that one specific mechanism does not endow resistance on its own; interplay of several defence systems is necessary. On the other hand, cell protection can be achieved in different ways so as to guarantee optimal survival. Acknowledgment Authors are very thankful to the Department of Science & Technology of Chandigarh administration for financial help for this work. References [1] B. Pscheidt, A. Glieder, Yeast cell factories for fine chemical and API production, Microb. Cell Fact. 7 (2008) 25. [2] P.J. Slininger, R.J. Bothast, J.E. Van Cauwenberge, C.P. Kurtzman, Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus, Biotechnol. Bioeng. 24 (1982) 371–384. [3] V.J. Higgins, A.G. Beckhouse, A.D. Oliver, P.J. Rogers, I.W. Dawes, Yeast genomewide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation, Appl. Environ. Microbiol. 69 (2003) 4777–4787. [4] H. Alexandre, V. Ansanay-Galeote, S. Dequin, B. Blondin, Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae, FEBS Lett. 498 (2001) 98–103. [5] T.N. Dinh, K. Nagahisa, K. Yoshikawa, T. Hirasawa, C. Furusawa, H. Shimizu, Analysis of adaptation to high ethanol concentration in Saccharomyces cerevisiae using DNA microarray, Bioprocess Biosyst. Eng. 32 (2009) 681–688. [6] E.N. Biriukova, A. Arinbasarova, A.G. Medentsev, Adaptation of the yeast Yarrowia lipolytica to ethanol, Mikrobiologiia 78 (2009) 189–194. [7] V. Costa, M.A. Amorim, E. Reis, A. Quintanilha, P. Moradas-Ferreira, Mitochondrial superoxide dismutase is essential for ethanol tolerance of Saccharomyces cerevisiae in the post-diauxic phase, Microbiology 143 (Pt. 5) (1997) 1649–1656. [8] M. Penninckx, A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses, Enzyme Microb. Technol. 26 (2000) 737–742. [9] M.J. Penninckx, An overview on glutathione in Saccharomyces versus nonconventional yeasts, FEMS Yeast Res. 2 (2002) 295–305.

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