Oxidative Stress Protection and Glutathione ... - Springer Link

Appl Biochem Biotechnol (2014) 174:2307–2325 DOI 10.1007/s12010-014-1188-4

Oxidative Stress Protection and Glutathione Metabolism in Response to Hydrogen Peroxide and Menadione in Riboflavinogenic Fungus Ashbya gossypii S. Kavitha & T. S. Chandra

Received: 29 March 2014 / Accepted: 19 August 2014 / Published online: 2 September 2014 # Springer Science+Business Media New York 2014

Abstract Ashbya gossypii is a plant pathogen and a natural overproducer of riboflavin and is used for industrial riboflavin production. A few literature reports depict a link between riboflavin overproduction and stress in this fungus. However, the stress protection mechanisms and glutathione metabolism are not much explored in A. gossypii. In the present study, an increase in the activity of catalase and superoxide dismutase was observed in response to hydrogen peroxide and menadione. The lipid peroxide and membrane lipid peroxide levels were increased by H2O2 and menadione, indicating oxidative damage. The glutathione metabolism was altered with a significant increase in oxidized glutathione (GSSG), glutathione peroxidase (GPX), glutathione S transferase (GST), and glutathione reductase (GR) and a decrease in reduced glutathione (GSH) levels in the presence of H2O2 and menadione. Expression of the genes involved in stress mechanism was analyzed in response to the stressors by semiquantitative RT-PCR. The messenger RNA (mRNA) levels of CTT1, SOD1, GSH1, YAP1, and RIB3 were increased by H2O2 and menadione, indicating the effect of stress at the transcriptional level. A preliminary bioinformatics study for the presence of stress response elements (STRE)/Yap response elements (YRE) depicted that the glutathione metabolic genes, stress genes, and the RIB genes hosted either STRE/YRE, which may enable induction of these genes during stress. Keywords CAT . SOD . LPX . GSH metabolism . mRNA levels . STRE/YRE

Introduction Ashbya gossypii is a natural overproducer of riboflavin and the first filamentous hemiascomycete fungus used in industrial large-scale riboflavin production [1]. Whether riboflavin overproduction is beneficial or toxic to A. gossypii has been an S. Kavitha : T. S. Chandra (*) Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India e-mail: [email protected] T. S. Chandra e-mail: [email protected]

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Appl Biochem Biotechnol (2014) 174:2307–2325

intriguing question. Riboflavin production was increased by nutrient limitation (nutrient stress) and decreased by cAMP supplementation (a negative stress signal) [2, 3]. In our previous study, we showed an increase in riboflavin production on supplementation of antioxidant vitamin E (VE) and oxidant menadione [4]. A recent study depicted Yap1-mediated induction of riboflavin when supplemented with H2O2 [5]. A. gossypii is a plant pathogen and has to deal with the plant defense mechanisms for survival. One important plant defense is oxidative burst which leads to production of reactive oxygen species (ROS), particularly H2O2 [5]. Therefore, it is now understood that riboflavin overproduction in A. gossypii constitutes a scavenging mechanism against the free radicals produced by plant defense [5]. Apart from overproduction of riboflavin, the fungus may host other antioxidant defense molecules to protect itself. This includes stress enzymes like catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX), which are known to increase during stress, as in Saccharomyces cerevisiae [6]. Exogenous supplementation of H2O2 and menadione leads to increased CAT, SOD, and GPX induction in Schizosaccharomyces pombe and Aspergillus niger [7, 8]. However, in A. gossypii, the study of crucial stress defense enzymes like CAT, SOD, and GPX under stress conditions is fragmentary. The present study aims at finding the role of these crucial enzymes, apart from riboflavin overproduction, in protection against H2O2 and menadione stress. The reduced glutathione (GSH) and GSH metabolic enzymes are also involved in protection against ROS apart from SOD and CAT in fungal species such as Penicillium chrysogenum [9]. The study of GSH metabolism is very important in A. gossypii organism, due to the following reasons: Under stress conditions, GSH is utilized to scavenge the ROS directly (Fig. 1) by donating a reducing equivalent (H++ e−) from its thiol group of cysteine to other unstable reactive oxygen species [10]. Riboflavin is also an antioxidant and free radical scavenger by itself [11]. Hence, glutathione and riboflavin may reciprocate in this riboflavin overproducer, and any significant findings of the study could be exploited for improving riboflavinogenesis. Secondly, riboflavin is the precursor for the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which participate in various oxidation and reduction reactions in the cells [12]. Glutathione reductase requires FAD as a cofactor along with NADPH for the reduction of oxidized glutathione to GSH (Fig. 1) [13]. Thus, riboflavin overproduction by A. gossypii could alter the GSH metabolism differently when compared to other filamentous fungi. In view of the above, the present study explored the oxidative stress response and GSH metabolism at the biochemical and molecular levels in the presence of peroxide and superoxide.

Materials and Methods Menadione, tetramethoxy propane (TMP), thiobarbituric acid (TBA), reduced and oxidized glutathione, glutathione reductase, dithio-bis-nitrobenzoic acid (DTNB), agarose, and ethidium bromide were procured from Sigma-Aldrich, IL, USA. 1-Chloro-2,4-dinitrobenzene (CDNB) and other chemicals used for the study were from Sisco Research Laboratories (SRL), Mumbai, India. The messenger RNA (mRNA) isolation kit, M-MulV RT-PCR Kit (Moloney murine leukemia virus), and DNA gel extraction kit were procured from Bangalore Genei, Bangalore, India. The complementary DNA (cDNA) sequencing was done from Bangalore Genei, Bangalore, India.


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Fig. 1 Possible influence of hydrogen peroxide, menadione, or riboflavin on the enzymatic and nonenzymatic components of GSH metabolism (compiled from Herrero et al. [6] and Masella et al. [13])

Organism, Growth Conditions, and Exposure to Stressors A. gossypii culture, NRRL Y-1056, was obtained from NCAUR, IL, USA. It was maintained on yeast-malt extract agar (YMA) slants of the following composition (g/l): 5 g peptone, 3 g yeast extract, 3 g malt extract, 10 g glucose, and 20 g agar. Mycelium was grown in Ashbya full medium (AFM) containing (g/l) 10 g casein, 10 g yeast extract, 20 g glucose, and 1 g myoinositol [14] at 180 rpm at 30 °C for all experiments. A preinoculum of 0.5 % grown for 48 h in the above medium was used to inoculate 50 ml of AFM in a 250 ml brown Erlenmeyer flask for all experiments. H2O2 (MW 34.01) was added fresh from a stock bottle of 30 % to obtain final concentrations of 10, 25, and 50 mM; 218 μl was added from a stock of 0.1 mg/ml of menadione (MW 376.23) to obtain a concentration of 2.5 μM. GSH (MW 307.3) was dissolved at concentrations of 154 mg/ml (stock 1) and 1.54 mg/ml (stock 2) in sterile water. From stock 1, 10 μl, 100 μl, and 1.0 ml were added to the AFM broth to get the final concentrations of 100 μM, 1 mΜ, and 10 mM, respectively. From stock 2, 40 and 100 μl were added to 50 ml of AFM broth to obtain 4 and 10 μM, respectively. Biomass was determined after drying the cells, harvested by centrifugation, to constant weight at 98 °C [4]. Total and extracellular riboflavin was estimated spectrofluorimetrically using the ISI standard procedure as described elsewhere [4]. A constant amount of frozen cells was used for cell-free extract (CFE) preparation using a mortar and pestle as mentioned previously [4].

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Analysis of Nonenzymatic Stress Parameters GSH was measured, in the protein free cell extract, by the formation of 5-thio-2-nitrobenzoic acid with DTNB spectrophotometrically at 412 nm as described earlier [4]. Protein was precipitated with 10 % TCA immediately after cell-free extract preparation and GSH measurements were performed. The total glutathione (GSH+oxidized glutathione (GSSG)) was measured after the GSSG present in the sample was converted to GSH by the highly specific glutathione reductase and NADPH. The total GSH was estimated similar to that of reduced GSH [4]. Lipid peroxidation (LPX) and membrane lipid peroxidation (MLPX) were assayed by their reaction with thiobarbituric acid (TBA) to form a colored adduct with a maximum absorbance at 532 nm. The quantification of the adduct formed was done by RP-HPLC using a RP-C18 Hibar column as described earlier [4]. Analysis of Enzymatic Stress Parameters The activity of SOD was measured as the inhibition of the rate of reduction of cytochrome c by the superoxide radical, formed by the xanthine-xanthine oxidase system. The reduction was observed at 550 nm by continuous spectrophotometric rate determinations. One unit inhibits the rate of reduction of cytochrome c by 50 % at pH 7.8; 0.1 ml of CFE (containing 1.0– 2.0 mg of protein/ml) was used for the assay [15]. The CAT activity was measured by the decrease in absorbance of H2O2 at 240 nm as described earlier [4]. The amount of reduced glutathione consumed for decomposition of hydrogen peroxide was measured for assaying the GPX activity as described [4]. Glutathione reductase (GR) was assayed by measuring the formation of NADP, which is accompanied by a decrease in absorbance at 340 nm. One unit of the enzyme is the oxidation of 1 μmol of NADPH/min at 25 °C at pH 7.0. CFE (0.1 ml containing 1.0–2.0 mg of protein/ml) was used for the assay [16]. The glutathione S transferase (GST)-catalyzed formation of GS-DNB (a dinitrophenyl thioether) was detected by a spectrophotometer at 340 nm. One unit of GST activity is defined as the amount of enzyme producing 1 μmol of GS-DNB conjugate/min; 0.3 ml of CFE (containing 1.0–2.5 mg/ml of protein) was used for the assay [17]. The protein content in the CFE was determined by the method of Lowry et al. using bovine serum albumin as standard [18]. mRNA Expression of the Stress Transcription Factors and Stress Genes The primers for genes were designed from the Ashbya Genome Database using “PRIMERBLAST” and “NET PRIMER” softwares (Table 1). The cells grown in the presence of H2O2 and menadione were harvested on days 2 and 3, and the total RNA was isolated followed by mRNA purification using the mRNA purification kit. The mRNA (90–100 ng) was reverse transcribed to cDNA followed by amplification using gene-specific primers. Multiplex PCR was carried out for amplification of cDNA of SOD1, GSH1, YAP1, and TEF in one tube and RIB3, CTT1, and MSN2 in another tube. All procedures were carried out after quantification and normalization of the RNA and DNA at each step. The PCR reactions were done at 30 cycles after ensuring that saturation is not attained at 30 cycles. The PCR products were electrophoresed by loading 15 μl of sample (100–200 ng/μl), 2 μl of 6× gel loading buffer, and 2 μl of ethidium bromide (0.5 μg/ml) in each lane. The bands were photographed using gel doc and intensity was quantified using Quantity One software. The PCR products were sequenced to confirm whether the primers used amplified the desired gene sequences. The experiments were repeated in three sets to confirm the results obtained.


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Table 1 Primer sequences designed for amplifying the gene of interest Group chosen for multiplex PCR

Gene / length (bp)

Forward primer (5′→3′)/length (from–to) (bp)

Reverse primer (5′→3′)/length (from–to) (bp)

Product length (bp)

Annealing temperature (°C)

Group 1

TEF / 1,377

GCCATCTTGATC ATTGCTGG / 334–353

TTGACTTCAGTGGT GACACC / 863–844

530

55

GSH1 / 1,986

GCGATCCGCT GATGTGGGGG / 125–144

ATGTTCGCCGTCAG CGTCGG / 572–553

448

59

YAP1p / 867

GACGGCGTGGGG AAGAAGGC / 422–441

CCGACATCCAGGCG GCGTTT / 794–775

373

59

SOD1 / 522

CTGCGGGCAGTTGC GGTTC / 61–79

CCGTGCTCCGTGTT GGGCTC / 191–172

131

59

RIB3 / 639

AAGCCCATGCAC TGATATCG / 6–25

GCAAGACCGTGCTT CTTGC / 590–572

585

55

CTT1 / 1,524

GCGGGTCGGCGAT GTTC / 260–276

CGGTACGACGCAGG GAT / 611–594

352

55

MSN2 / 1,716

TGGGTCTGACAGGG GCG / 65–81

GCGTGTTCGGCGTG TCCG / 175–159

111

55

Group 2

Ashbya Genome Database

Analysis of Stress Response Elements and Yap Response Elements on the Promoter Region of Stress Genes and RIB Genes The upstream 1,000 bp of the genes along with the open reading frame (ORF) was analyzed for the presence of sequences of stress response elements (STRE) (5′-AGGGG-3′, 5′-CCCCT3′) and Yap response elements (YRE) (5′-TTA(C/G)TAA-3′) using the Needleman-Wunsch global sequence alignment tool (http://blast.ncbi.nlm.nih.gov/). The other YRE motifs analyzed were 5′-TGACTAA-3′ and 5′-TGACTCA-3′. In the present study, the presence of a single sequence with 100 % identity to the STRE and YRE in the upstream 1,000 bp was considered as a response element. Statistical Analysis ANOVA and paired t test were used for statistical significance analysis. p values less than 0.05 were considered significant. The values represented are means of three independent experiments.

Results A time course of A. gossypii showed maximum biomass on days 2 and 3 followed by autolysis on day 4 (Fig. 2, control). The total riboflavin production increased from day 2 and reached maximum on day 3, after which it remained constant. The extracellular riboflavin increased progressively from day 2 and kept increasing throughout the time course. However, the increase in extracellular secretion on days 4 and 5 could be attributed to the autolysis of the cells. Due to the above reasons, most of the further experiments were performed only on days 2 and 3 of growth.


Extracellular riboflavin mg/l

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Control 10mM 25mM 50mM

300 250 200 150 100 50 0

Total riboflavin mg/l

400 350 300 250 200 150 100 50 0 14

Dry Biomass g/l

12 10 8 6 4 2 0 0

1

2

3

4

5

Days

Fig. 2 Effect of the addition of different concentrations of hydrogen peroxide on biomass and riboflavin production

Effect of Different Concentrations of H2O2 on Biomass, Total Riboflavin, and Extracellular Riboflavin The abovementioned parameters were studied with different levels of H2O2 to find the concentration at which H2O2 influenced riboflavin production without compromising the biomass. Hydrogen peroxide was used in three different concentrations (10, 25, and 50 mM) to explore the effect on biomass and riboflavin production as a time course (Fig. 2). The biomass was not affected with 10 and 25 mm H2O2 and was decreased with 50 mM. H2O2 at 25 mM increased riboflavin production by 1.6-fold on day 2 and 1.7-fold on day 3 and was chosen for further experiments on stress response and GSH metabolic studies due to the following reasons: Since 25 mM was not toxic, it is understood that the cells could mount an effective stress response at this concentration. Apart from this, 25 mM had increased riboflavin production and thus is appropriate for GSH metabolism studies. In an earlier study, menadione (2.5 μM), a superoxide generating agent, also increased riboflavin production (207±7.1 mg/l) and secretion (76.2±1.7 mg/l) compared to controls without affecting biomass (Fig. 3) [4]. Menadione (2.5 μM) was used in the present study to compare its effects with hydrogen peroxide.


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Fig. 3 Effect of different concentrations of menadione on biomass and riboflavin production [4]

Effect of H2O2 and Menadione on Oxidative Stress Parameters (a) Catalase and Superoxide Dismutase Enzyme Activities The activities of CAT and SOD were measured as a time course for 5 days in controls and on supplementation of H2O2 (25 mM) and menadione (2.5 μM) (Table 2). Though analysis was done on all days, the results of days 2 and 3 are discussed, since autolysis ensues from day 4. H2O2 and menadione significantly increased CAT and SOD on days 2 and 3 compared to controls. Menadione predominantly increased SOD, with the highest value on day 2 (6.2±0.16 U/mg). There was a preferential increase in CAT activity on H2O2 supplementation and the highest value was recorded on day 2 (428± 28.8 U/mg) (Table 2). (b) Lipid Peroxide and Membrane Lipid Peroxide Formation Lipid peroxidation indicates damage to lipids caused by oxidative stress. In the present study, lipid peroxidation measurements were used as a marker to confirm oxidative stress in the cells. LPX and MLPX were significantly increased on days 2 and 3 by H2O2 and menadione (Table 3), indicating oxidative damage, which could not be prevented, by an increase in SOD and CAT (Table 2).

a&

a$

2.2±0.12 2.7±0.10a$

2.5±0.30

2.5±0.12 2.3±0.19a&

3.2±0.12

b$

4.4±0.37

a$

3.5±0.15

b#

(1.3)

(1.26)

5.1±0.20a@ (1.0)

4.9±0.50a@

a#

H2O2

Control

Superoxide dismutase (U/mg protein)

2.2±0.15 2.3±0.15a$

a$

4.9±0.20

c@

(2.0)

6.2±0.16 (1.7)

c#

5.3±0.15a@ (1.0)

Menadione

a$

116±0.86 19.4±0.66a&

110±6.5

a$

294±13.6

a#

229±23.7a@

Control

Catalase (U/mg protein)

158±1.7 17.2±1.7a&

c$

155±1.2

b$

428±28.8

(1.45) (1.4)

b#

370±11.4a@ (1.6)

H2O2

158±4.5c& 32.3±1.5b*

193±9.6c$ (1.75)

367±20.4c# (1.2)

217±3.7a@ (0.94)

Menadione

Values (n≥3±SD). ANOVA. p value