Gene 535 (2014) 131–139

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Differential gene expression in Giardia lamblia under oxidative stress: Significance in eukaryotic evolution Dibyendu Raj a,1, Esha Ghosh a,1, Avik K. Mukherjee a, Tomoyoshi Nozaki b, Sandipan Ganguly a,⁎ a b

Division of Parasitology, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

a r t i c l e

i n f o

Article history: Accepted 20 November 2013 Available online 7 December 2013 Keywords: Giardia Oxidative stress Transcriptomic

a b s t r a c t Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 μM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production. To study differential gene expression under three types of oxidative stress, a Giardia genomic DNA array was constructed and hybridized with labeled cDNA of cells with or without stress. The transcriptomic data has been analyzed and further validated using real time PCR. We identified that out of 9216 genes represented on the array, more than 200 genes encoded proteins with functions in metabolism, oxidative stress management, signaling, reproduction and cell division, programmed cell death and cytoskeleton. We recognized genes modulated by at least ≥2 fold at a significant time point in response to oxidative stress. The study has highlighted the genes that are differentially expressed during the three experimental conditions which regulate the stress management pathway differently to achieve redox homeostasis. Identification of some unique genes in oxidative stress regulation may help in new drug designing for this common enteric parasite prone to drug resistance. Additionally, these data suggest the major role of this early divergent ancient eukaryote in anaerobic to aerobic organism evolution. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Giardia lamblia, a biflagellate gastro-intestinal parasite in Diplomo nadida causes giardiasis that affects 300 million people worldwide (Ortega and Adam, 1997). Giardia is a microaerophillic organism and does not usually tolerate elevated oxygen level. In the upper intestinal lining, where this organism generally resides, the O2 concentration has

Abbreviations: ROS, reactive oxygen species; NADH, (reduced) nicotinamide adenine dinucleotide; PCD, programmed cell death protein like protein; TYIS-33, tryptone–yeast extract–iron–serum-33; PBS, phosphate buffered saline; PCR, polymerase chain reaction; HEPES, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid; NADPH, (reduced) nicotinamide adenine dinucleotide phosphate; ATP, adenosine tri-phosphate; VSP, variant specific surface protein; SOD, superoxide dismutase; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; DCF, 2′,7′dichlorofluorescein; SSC, standard saline citrate; dUTP, deoxyuridine triphosphate; dTTP, deoxythymidine triphosphate; RT-PCR, real time PCR; GSH, glutathione; SRP-64, signal recognition particle-64; PCV, protein for cell viability. ⁎ Corresponding author. Tel.: +91 33 2363 3855. E-mail address: [email protected] (S. Ganguly). 1 Authors have equal contribution. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.048

been measured at 60 μM (Atkinson, 1980). On the other hand Giardia is capable of tolerating 0–50 μM dissolved O2 (Lloyd et al., 2000). The detailed mechanism, by which the parasite could aid in the detoxification of reactive oxygen species (ROS) produced during an oxidative stress, is still not clear. In addition to this, some of the conventional enzymes of detoxifying ROS, such as superoxide dismutase (SOD), catalase, peroxidase, glutathione, glutathione reductase are absent in G. lamblia (Brown et al., 1995). However, it possesses a prokaryotic H2O-producing NADH oxidase, a membrane associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulfide reductase and the low molecular weight thiols, L-cysteine, thioglycolate, sulfite and coenzyme-A (Brown et al., 1998). Cysteine is the major low molecular weight thiol in G. lamblia and reduced thiols serve as a defense against oxidative stress and as a mechanism to maintain a reduced intracellular environment. L-Cysteine has antioxidant properties, and is used for biosynthesis of glutathione, which is found in most eukaryotes including humans. Oxidative stress triggers a range of physiological, pathophysiological, and adaptive responses in cells either as a result of cellular damage or

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through a specific signaling molecule. These responses ultimately modulate transcriptional outputs to influence cell fate and disease processes. In the past two decades, a number of transcription factors and signaling pathway have been identified and delineated to mediate critical transcriptional responses to oxidative stress. These examples demonstrate the importance as well as the complexity of how alterations in intracellular ROS are converted into discrete and reproducible alterations in gene expression. Recently, it has become possible to decipher transcriptional programs of an organism by studying gene expression en masse (Brown and Botstein, 1999). Differences in cell types or states are correlated with changes in the mRNA levels of many genes (DeRisi et al., 1997). DNAmicroarray technology provides an opportunity to look simultaneously at changes in gene expression in thousands of genes under different physiological conditions (DeRisi and Iyer, 1999). In the present study, we performed DNA microarray analysis of gene expression in stressed G. lamblia trophozoites by H2O2, metronidazole and also given stress with a cysteine–ascorbate deprived medium. The differentially regulated genes fall into five groups of functionally related proteins. These functional categories are: (i) metabolic enzymes; (ii) structural proteins; (iii) kinases and phosphatases; (iv) cell cycle and proliferation controller; and (v) cell death regulators. 2. Materials and methods 2.1. Maintenance of cultures G. lamblia trophozoites were maintained in TYIS-33 medium that was supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% adult bovine serum, according to the methods of Diamond et al. (1978). All of the experiments were conducted with trophozoites that had been harvested during the logarithmic phase of growth. 2.2. Activation of trophozoites with oxidative stress

30, 60, 80, and 120 s by use of a nebulizer (Invitrogen) which was connected to the laboratory's compressed-air line. The sheared DNA was blunt-end repaired by use of T4 DNA and Klenow polymerases, and the ends were dephosphorylated by using calf intestinal phosphatase, to create a suitable substrate for TOPO cloning (Invitrogen). DNA fragments were ligated into PCR4 Blunt-TOPO vector (Invitrogen). The cloning reaction was performed for 5 min at room temperature, with undiluted and with 3- and 9-fold serially diluted blunt-end DNA; 3.3 ml of precipitated TOPO cloning reaction was electroporated into 50 ml of One Shot TOP10 Eletrocomp Escherichia coli (Invitrogen), and the transformants were incubated overnight on agar plates in the presence of X-gal and 100 mg/ml ampicillin. To check the quality, specificity and integrity of the genomic DNA library, ~250 clones were randomly chosen and sequenced (data not shown). Gene repetition and fragmentation were at minimum level. 2.4. G. lamblia microarray Individual white colonies from the fresh random-fragment genomic DNA library were transferred to a 600 μl solution of Luria–Bertani medium (with 100 μg/ml ampicillin) in individual wells of a 96-well plate and were incubated for 18 h at 37 °C; 1 μl from each well was used for amplification of the insert by PCR, using the M13 forward and reverse primers. After analysis of the PCR products on 1% agarose gels, the products were consolidated onto 96-well plates. The PCR products from the 96-well plates were precipitated, washed, and transferred to 384-well plates and were printed on the aminisilane-coated glass slides, by use of the GeneMachine®. The concentration of each DNA product in 3× standard saline citrate (SSC: 1× SSC is 0.15 mol/l NaCl, 0.015 mol/l sodium citrate) printing solution was 200 ng/μl, which produced acceptable spot quality. After the DNA solution was deposited on every slide, the tips were washed with 0.5× SSC and dried, and the process was repeated for the next set of DNA samples, with the new spots offset a small distance (200 mm) relative to previous spots, to produce a high-density grid. Microarrays were printed under controlled environmental conditions (i.e., a temperature of 19 °C and a relative humidity of 50%). At the end of the print run, the slides were allowed to dry for ~ 12 h.

In the present study, three conditions have been chosen to generate oxidative stress in the trophozoites in vitro. First, hydrogen peroxide (H2O2) is a very well known chemical reagent that can generate free oxygen radicals very easily (Lindley et al., 1988). Secondly, metronidazole is a commonly used drug against giardiasis in most developing countries. Its mode of action involves free nitro radical generation within the organism (Brown et al., 1998). The third, is the modified medium that is devoid of cysteine and ascorbate. Cysteine has been found to protect G. lamblia trophozoites from thiol-blocking reactants, indicating a role as a reducing agent for the protection of crucial thiol groups. Ascorbic acid also protects the trophozoites under high PO2 (Tekwani and Mehlotra, 1999). Dose and time kinetics of the three different oxidative stress generating conditions has been standardized following the IC50 values according to Lindley et al. (1988) and Sadhu et al. (2004). Finally, from standardized data, 0.1 μM H2O2 and 1 μg/ml metronidazole have been administered to all the experiments mentioned below. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA) assay was performed to observe the ROS generation in each of the above cases. Briefly, 5 × 106 trophozoites of Giardia for each set (control and three oxidative stressed sets) were collected and dissolved in PBS. 2 μl of DCFDA (stock 100 μM) was added in each case. Then all the sets were incubated for 15 min at 37 °C in the dark. The samples were washed thrice and finally slides were prepared for confocal study.

Total RNA was isolated from ~ 5 × 107 trophozoites by use of a TRIZOL kit (Invitrogen) and as directed by the manufacturer. Optical density readings were taken at 260 nm and 280 nm. The integrity of total RNA was checked in denatured formaldehyde gel, according to standard protocol (Sambrook et al., 1989). Fluorescently labeled cDNA copies of the total RNA pool were prepared from both oxidative stress-induced and non-induced G. lamblia. In brief, 30 μg of total RNA was reverse transcribed by using Stratascript reverse transcriptase (Stratagene), 10 μg/μl of oligo-dT 0.1 M dithiothreitol, and a 3:2 ratio of amino-allyl dUTP to dTTP, for 2 h at 42 °C. After hydrolysis of the RNA, the samples were purified by the use of a Qiagen® column. The monofunctional NHS-easte Cy-3 and Cy-5 dyes were coupled with the cDNA, and the unincorporated/quenched Cy-dyes were removed by the use of a column; the Cy-5 labeled sample was eluted into the tube containing the corresponding Cy-3 labeled sample. The two cDNA pools to be compared were mixed and were applied to the array in a hybridization mixture containing 3× SSC, 25% SDS, and 25 mmol/l HEPES (Ph 7.0). Hybridization took place in the Hybstation from Genomic Solutions® by setting a standardized protocol.

2.3. Genomic shotgun library

2.6. Data acquisition, analysis and sequencing

Genomic DNA was isolated from 108 G. lamblia trophozoites using the method of Huber et al. (2001). RNA contamination was removed by RNase digestion for 30 min at 37 °C. Genomic DNA was sheared for

Arrays were scanned by use of ScanExpressHT (Perkin-Elmer); images were acquired by scanning with the Cy3 and Cy5 channels at a resolution of 5 mm. The Cy5/Cy3 fluorescence ratios and log10-transformed ratios

2.5. RNA isolation, labeled cDNA preparation, and microarray hybridization

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Fig. 1. Standardization of in vitro stress generation. A. Dose and time kinetics under metronidazole treatment. B. Dose and time kinetics under H2O2 treatment. C. Growth and time kinetics under modified medium treatment.

were calculated from the normalized values. Genes whose expression changed N2-fold relative to those in control G. lamblia were selected for further analysis. Individual clones were sequenced by the use of anautomated ABI Prism 3100 genetic analyzer. These sequences were compared with others through searches of the DNA and proteinsequence databases at the National Centre for Biotechnology Information (Bethesda, MD), by use of the BLAST program. Each experimental set has been repeated twice and checked by dye swapping.

2.7. Validation by real time PCR (RT-PCR) Based on the importance of biochemical and molecular biological activities, 34 genes (identified from the transcriptomic result) were chosen for further confirmation of their differential expression by real time PCR. Suitable primers for the 34 genes have been designed, a housekeeping gene (here Actin) has been normalized and the amount of cDNA for RT-PCR has been standardized before starting the validation by real time. PCR was started by initiating the Taq polymerase reaction at 95 °C (15 min). Subsequent DNA amplification was performed in 40 cycles including denaturation (94 °C for 15 s); annealing (60 °C for 30 s); and extension (72 °C for 30 s). For statistical analysis, three independent experiments were performed (Muller et al., 2008). Comparative Ct method has been adopted to analyze the real time data. The RT-PCR primer used in this study has been mentioned in the Supplementary Table 1.

3. Results and discussion 3.1. Standardization of in vitro oxidative stress generation We have standardized oxidative stress generation and reactive oxygen species production in all the three cases viz. H2O2 application, metronidazole treatment and incubation with modified medium. As we have seen, with increasing concentrations of H2O2 and metronidazole the rate of cell (trophozoites) death increased (Figs. 1A and B). Time kinetics and growth kinetics of the trophozoites have been shown that the trophozoites of G. lamblia incubated with cysteine–ascorbate deprived medium, lost their viability and were dislodged from the surface of the glass tubes at a greater rate than the trophozoites that were incubated with normal TYIS-33 medium (Fig. 1C). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA), was used to detect the intracellular generation of reactive oxygen intermediates in Giardia trophozoites by using a confocal microscope. H2DCFDA was added to the suspension of G. lamblia (5 × 106 cells/ml) at a final concentration of 5 μM. We observed that trophozoites, treated with H2O2, metronidazole and cysteine–ascorbate deprived medium have greater fluorescence than the non-treated trophozoites (Fig. 2). The green fluorescence within the cells indicates that the cell-permeable and nonfluorescent 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), also known as dichlorofluorescein diacetate, upon cleavage of the acetate groups by intracellular esterases and oxidation, is converted to the highly fluorescent 2′,7′-dichlorofluorescein (DCF).

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Fig. 2. DCFDA assay: of G. lamblia H2DCFDA-loaded cells. Fluorescence was monitored from suspensions of live cells. A. Control cells (without stress) with no significant ROS generation, hence green fluorescence is absent. B. Stressed cells with H2O2 treatment. C. Stressed cells with metronidazole treatment. D. Stressed cells with modified medium treatment: sufficient amount of reactive oxygen particles are present, hence, on oxidation, the nonfluorescent H2DCFDA is converted to the highly fluorescent dichlorofluorescein (DCF).

3.2. Array analysis and RT PCR validation The hybridized slides were scanned using ScanArray® software in the scanner and more than 200 clones were identified that show 2 folds or higher times up-regulation or down-regulation than the control set (full data not shown). The result was cross checked twice and also by dye swapping. The clone numbers that matched in all the cases have been chosen for further sequencing analysis. The major steps of genomic DNA library preparation and microarray construction i.e. Genomic DNA isolation, shearing, amplification and purification are depicted (Fig. 3). To well understand the role of oxidative stress in transcriptional regulation of gene expression in G. lamblia, we performed analysis of genome wide gene expression upon three different oxidative stresses. We identified some genes significantly regulated under oxidative stress and is categorized according to their predicted functions (Table 1). From the array analysis some interesting gene candidates were selected for real time PCR validation (Figs. 4A, B, C). Transcriptional regulation due to oxidative stress shows that it affects the parasite cell and can change several physiological activities. This has helped to generate basic knowledge about some pathways controlling the survival and evolution of this human enteric parasite. 3.3. Oxygen metabolism It has long been established that Giardia follows anaerobic bacteria metabolism (Brown et al., 1998) and it indicates its primitive eukaryotic

nature. NADH oxidase provides a means of removing excess H+ producing H2O. Giardia produces energy by carbohydrate and amino acid fermentation in the cytoplasm. But very recently, proteome analysis of Giardia mitosome (reduced mitochondria) has shown that it contains NADH oxidase, thioredoxin reductase, ferredoxin, peroxiredoxin etc. (Jedelsky et al., 2011). Hence cytoplasmic energy generation of Giardia has become doubtful. In our study the transcriptomic profile of this parasite under oxidative stress shows up-regulation of NADH oxidase along with some other enzymes like NADPH oxidoreductase, disulfide reductase, peroxiredoxin, dihydrogenase etc. Pyruvate-ferredoxin oxidoreductase and dehydrogenase have not been found in the mitosomal protein profile (Emelyanov and Goldberg, 2011) and proved to be present in the cytoplasm. Hence oxygen metabolism under oxidative stress is controlled by both cytoplasmic and mitosomal enzymes in Giardia (Table 1). Super oxide dismutase is absent in Giardia, hence the fate of a super oxide radical was unknown previously. But few years back a non-heme iron protein (putative neelaredoxin?) has been reported (Upcroft and Upcroft, 2001) and in Giardia assemblage E, a neelaredoxin like enzyme, GIP 15_1799 (superoxide reductase) has been found. In case of assemblage A, 9 sequences have been identified among which CH991785 is showing 91% homology [source: GiardiaDB]. In G. lamblia, O2 is directly converted to H2O by NADH oxidase. Pyruvate ferredoxin oxidoreductase converts oxidized ferredoxin to a reduced one. Malate dehydrogenase is also upregulated during oxidative stress. Disulfide bonds are reduced by disulfide reductase. Superoxide dismutase is absent in Giardia but a non-heme iron protein (putative neelaredoxin)

Fig. 3. Major steps of genomic DNA library and array construction. A. Genomic DNA isolation from Giardia lamblia. B. Genomic DNA shearing between 1 and 2 kb sizes. C. PCR amplification of the recombinant DNA. D. Purified PCR product for microarray spotting.

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Table 1 Identification of oxidative stress induced gene expression in the microarray. Names of the genes

Gene ID

Up/down regulationa H2O2

Metabolic enzymes coding genes NADH oxidase GL50803_9719 NADH ferredoxin oxidoreductase GL50803_17151 Pyruvate ferredoxin oxidoreductase GL50803_114609 Thioredoxin reductase GL50803_9827 Nitroreductase GL50803_15307 Arginine deiminase GL50803_112103 Malate dehydrogenase GL50803_3331 Alcohol dehydrogenase GL50803_13350 Phosphatase and kinase coding genes Kinase GL50803_15548 CAM kinase GL50803_16034 NIMA related kinase GL50803_87945 POLO like kinase GL50803_104150 Serine threonine protein phosphatase Gl50803_21498 Protein phosphatase regulatory subunit A GL50803_16567 Transcriptional/translational protein coding genes Small subunit rRNA GL50803_r0019 Large subunit rRNA GL50803_r0013 Ribosomal protein L8/L2 GL50803_16086 TAR RNA loop binding protein GL50803_32741 Nuclear LIM interactor interacting factor-I GL50803_14905 TMP 55 GL50803_137641 Protein 21.1 GL50803_13590 Cell cycle regulatory, cell divisional and cell death deciding protein coding genes FtsJ cell division protein GL50803_16993 Spindle pole protein GL50803_8512 Homologous pairing (HOP) gene GL50803_4084 Disrupted meiotic cDNA (DMC) 1 GL50803_13346 PCD protein like proteinb GL50803_2933 b Protein for cell viability GL50803_6211 Structural proteins coding genes Beta giardin GL50803_4812 Dynein light chain GL50803_7578 Dynein heavy chain GL50803_101138 Some other important protein coding genes Hsp70B2 cytosolic form GL50803_88765 Hsp90 alpha GL50803_98054 Stress induced phosphoprotein (?) GL50803_27310 Cysteine rich variant specific protein GL50803_113297 Sodium–hydrogen exchanger III GL50803_102647 Cathepsin B precursor GL50803_17516 Clathrin heavy chain GL50803_102108 Tubulin specific chaperone E GL50803_16535 Midasin MDN1 GL50803_39312 Pseudouridylate synthase I GL50803_28831

4 2 3 3

Metronidazole

Modified medium

3 3.5 3.5

4.5 4 2.5 6

–

– −3.5 2 2

6 −4.5 4 2.5

–

−2 3 5 3 5 2.5

–

−3.5 5 4.5 3 4 –

2 2 3

3.5 4 4 5.5 2 2.5 2

−5 −3.5 −4.5 −4 2.5 –

4 4 3.5 2.5 3.5 2.5 4

–

– 3 2 3

4 3 3

2 2.5 – 2.5 −4 −3 −4 −3 2 –

4 2 4 4 – 3.5

4 3.5 2

4.5 4 2.5

5.5 4 2.5

6 3 4 2 2 2.5 5 3.5 −2 3.5

5 2.5 5

4.5 3.5 4 5 3 −2 2.5 2.5 – 4

– 3 3 3 5 −2.5 3

List of genes differentially regulated (more than 2 fold) in transcriptomics study during oxidative stress. a This data set is expressed as how many times the experimental genes are up-regulated or down-regulated with respects to the control set i.e. the un-induced normal one. b These two genes' expressions are highly variable with time. A time kinetics study has proved that PCD is down-regulated in the first 3–3.5 h in H2O2 & metronidazole and up-regulated after 3 h where it is just the reverse in the case of modified medium treated sets.

like enzyme has been found to be upregulated that may convert superoxide radicals to peroxides which then produce water by peroxiredoxin. Nitro-radicals generated during metronidazole application are detoxified by nitroreductases (Fig. 5). 3.4. Oxidative stress management This issue was thought to be controlled mostly by NADH oxidase. But from the transcriptomic result we see here that oxidative regulation is not only controlled by some metabolic enzymes, but also different types of heat shock proteins, sodium hydrogen exchangers, and some cys-rich proteins take a significant role in ROS detoxification. Oxidation of \SH bonds to disulfide formation are one of the very essential mechanisms which help to reduce reactive oxygen species. To prevent oxidative damage to oxygen-labile electron transport components, Giardia contains cysteine and disulfide reductase, the equivalent of the GSH system of higher eukaryotes, which serve as intracellular reductants to maintain a reduced intracellular environment. It was

observed that disulfide reductase was highly up-regulated during oxidative stress. Interestingly, a stress induced phosphoprotein has been found to be up-regulated. Along with that some kinases and phosphatases have been found to be differentially regulated during this time (Ma, 2010). Hence oxidative stress regulation in Giardia is a better term for oxidative stress management due to the involvement of several types of enzymes and proteins. 3.5. Alternative energy source Giardia can grow in the culture medium devoid of glucose and supplemented with amino acid sources (Edwards et al., 1992; Schofield et al., 1990). The formation of a high intracellular concentration of alanine is essential for parasite survival during changes to an environment of the type that it might be expected to experience during a passage of food and water along the host GI tract (Brown et al., 1998). Enzymes for the arginine dihydrolase pathway, especially arginine deiminase, have been observed to be up-regulated under cysteine–ascorbate deprived

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Fig. 4. Comparison of the G. lamblia genes modulated upon hydrogen peroxide stress, metronidazole stress, and cysteine-ascorbate deprived medium stress: A. Gene expression (fold change) under H2O2 stress. B. Gene expression (fold change) under metronidazole treatment. C. Gene expression (fold change) upon cysteine–ascorbate deprived medium treatment. (Gene abbreviation used: metabolic enzymes: NADHOX—NADH oxidase, NADHOR—NADH oxidoreductase, PFOR—pyruvate-ferredoxin oxidoreductase, DSRD—disulfide reductase, NR—nitroreductase, POR—peroxiredoxin, ALCDH—alcohol dehydrogenase, MALDH—malate dehydrogenase, ARGD—arginine dihydrolase. Structural proteins & others: BGDN—β giardin, DLC—dynein light chain, CHC—clathrin light chain, CMBP—cysteine rich membrane binding protein, NAHE—Na–H exchanger, HSP—heat shock protein, SIP—stress induced phosphoprotein. Kinases, cell divisional & meiotic proteins: CAMK—CAM kinase, PLK—polo-like kinase, NRK—NIMA related kinase, STP—serine threonine phosphatase, FCDP—Fts-J cell divisional protein, SPP—spindle pole protein, HOP—homologous pairing, DMC—disrupted meiotic protein.

medium treatment, revealing that activation of alternative energy metabolism produces additional ATP to maintain further cellular activities. Giardia can produce ATP faster by arginine utilization than from glycolysis

(Schofield et al., 1992). However, a gene encoding arginine deiminase was slightly down-regulated during hydrogen peroxide and metronidazole treatment. Down-regulation of the gene may partially contribute to the

Fig. 5. Model representations of metabolic enzymes working under oxidative stress. General scheme of metabolic enzymes involved in oxygen metabolism in Giardia lamblia.

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overall decrease in the metabolic flux across the arginine dihydrolase pathway. 3.6. Reproduction and cell division It was predicted that in response to stress, protomitochondrial symbionts released cytochrome c and up-regulated the reactive oxygen species (ROS) production of a host. This triggered the sexual recombination of the unicellular host and the formation of novel host genotypes, potentially leading to a less stressful environment for the protomitochondria (Blackstone and Green, 1999). Giardia usually undergoes asexual reproduction (binary fission) but the presence of sexually reproductive genes has been found during its genome sequencing. Some genes have been observed to be up-regulated when the trophozoites were exposed to the modified medium devoid of cysteine–ascorbate. These proteins are deeply involved in cell cycle regulation, cell division and reproduction. FtsJ cell division protein, spindle pole protein, homologous pairing protein and disruptive meiotic chromosomal protein have been found to be up-regulated under oxidative stress among these genes, of which the last two definitely indicate sexual reproduction and proving the hypothesis previously stated. Genes controlling the meiotic like pathway have also been observed to be regulated during encystations (Melo et al., 2008), though sex in Giardia has been referred to be rare, furtive or cryptic (Birky, 2009). 3.7. Mode of cell death It was hypothesized that the origin of eukaryotic programmed cell death is a consequence of aerobic metabolism (Frade and Michaelidis, 1997). In the previous paragraph, a hypothesis by Blackstone and Green (1999) was discussed partly. According to them, in metazoans, the mechanism of apoptosis involving cytochrome c may be a vestige of the process where programmed cell death is triggered instead of sexual reproduction. In another study, we reported a protease independent programmed cell death in Giardia under oxidative stress (Ghosh et al., 2009). When oxidative stress exceeds the limit of the parasite's tolerance level, the cells commit suicide. In the dying cells, a protein named programmed cell death protein has been found to be upregulated. A time-kinetics has been done where the increased expression of this protein has been observed. This protein is a type of phosphatase. Another protein named protein required for cell viability is inversely regulated with the previous one i.e. when the cells undergo death phase, expression level of this protein gradually decreases. Cathepsin B precursor gene has also been observed to be regulated under cell death during oxidative stress. Cysteine protease has been found to be down-regulated in the initial phase whereas, signal recognition

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particle-64 (SRP-64) gets up-regulated in the primary stage but gets down-regulated during the commencement of cell death (Fig. 6). The total cell death regulation under oxidative stress has been found to be a unique feature of this parasite. In our previous study, we obtained some results on the changes in parasitic cell cycle, cell morphology and cell death under oxidative stress. Transcriptomic data from the real-time PCR study has further elaborated that concept indicating the name of some significant genes regulating cell death of this parasite during oxidative stress.

3.8. Significance in evolution The appearance of eukaryotic cells ~ 2 billion years ago has been linked to the introduction of oxygen in the atmosphere and therefore aerobic metabolism (Dismukes et al., 2001). The increased presence of oxygen produces a more efficient energy source in the form of aerobic metabolism, producing 16–18 times more adenosine triphosphate (ATP) per hexose sugar than anaerobic metabolism (Dismukes et al., 2001). Since aerobic metabolism generates more energy, approximately 1000 more reactions can occur than under anaerobic metabolism (Dismukes et al., 2001). This allowed the generation of the new metabolites, for example, steroids, alkaloids and isoflavonoids (Jiang et al., 2010) and polyunsaturated fatty acids which are important elements of membranes; thus they must have been involved in promoting organelle formation and cell compartmentalization (Thannical, 2009). Since these appear important in superior eukaryotes, it has been hypothesized that such events influenced by increased oxygen levels, have influenced biological evolution. These examples demonstrate the importance as well as the complexity of how alterations in intracellular ROS are converted into discrete and reproducible alterations in gene expression. As Giardia, although with free-living relatives, were hypothesized to be representatives of the earliest eukaryotes, primitive phagocytes that had never acquired mitochondria, but which survived in marginal niches. But a closer scrutiny of the cell structure (doublemembrane structure derived from mitochondria known as mitosome) and its genome (which contain genes derived from mitochondria) betrayed the fact that these apparently primitively amitochondriate groups had once possessed mitochondria, but had later lost them in the course of specializing into anaerobic environments (Van der Giezen, 2009). Giardia has also a very important standpoint in evolutionary studies as a primitive eukaryote. Its biochemical and metabolic pathways can offer a good reply to several unanswered questions till date. Eukaryotes allocate more communication roles to proteins than prokaryotes, allowing for more complex and variable signaling pathways during oxidative stress. In order to achieve this, various changes in protein structure and function had to occur during evolution.

Fig. 6. Expression kinetics of cell death regulating proteins upon oxidative stress in Giardia lamblia. X axis: name of the gene; Y axis: FOLD change. PCD: programmed cell death protein like protein, GRP: glucose regulated protein 94 kDa (anti-apoptotic death regulator), CB5: cytochrome b5, CP: cysteine protease, CBP: cathepsin B precursor, PCV: protein for cell viability, SRP68: signal recognition particle-68.

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Fig. 7. A schematic diagram representing the role of oxidative stress behind eukaryotic evolution. Oxidative stress regulates different cell biological activities in this primitive organism to achieve evolution of higher eukaryotes with aerobic metabolism, sexual reproduction and programmed cell death.

As we have observed, oxidative stress is regulating different cell biological activities in this primitive parasite, thus evolution of higher eukaryotes with aerobic metabolism, sexual reproduction, and programmed cell death under higher oxygen tension (Fig. 7) can be studied further in detail. 4. Conclusion This study represents the first genome-wide analysis of transcriptional changes induced by hydrogen peroxide, metronidazole and also by L-cysteine–ascorbate deprived medium in G. lamblia. In the three experimental conditions it has been observed that different types of stress generating conditions regulate the stress management pathways differently. Some genes like heat shock proteins, NADH oxidase, pyruvate ferredoxin oxidoreductase etc. are always up-regulated during oxidative stress. But some genes are very specifically regulated depending on the mode of oxidative stress generation. Nitroreductase is much more upregulated in metronidazole stress than in other two conditions. Similarly, disulfide reductase is most upregulated in a modified medium. Structural protein like β-giardin is also most up-regulated in a modified medium than the other two. It has also been observed that some genes controlling giardial morphology are differentially regulated during stress in a modified medium. NADH oxidase, NADH ferredoxin oxidoreductase, pyruvate ferredoxin oxidoreductase, thioredoxin reductase, nitroreductase, arginine deiminase, alcohol dehydrogenase etc. have been found to make a good network for oxidative stress management. This also indicates that several pathways are involved in this situation as the enzymes are the representatives of some important biochemical pathways in other eukaryotes. In addition to this, a good number of hypothetical proteins are there which indicate their significant role in stress regulation requiring a wide scope for further studies on these unknown proteins. Some other proteins like Hsp90, β-giardin, cysteine rich VSPs, CAM kinase, serine threonine protein phosphatase etc. also play some important roles in Giardia survival. In the later section of this study, some genes have been proved to be regulated during cell death using quantitative real time PCR.

Programmed cell death protein like protein, cytochrome, cathepsin etc. are playing a significant role in cell death determination whereas GRP98 and protein for cell viability (PCV) are antiPCD and their expressions are inversely proportional with commencement of cell death. The present study has identified various biological processes involved in the regulation of gene expression pertaining to oxidative stress management in G. lamblia. These processes required several components, such as regulatory elements, transcription factors and cofactors, chromatin modification proteins and proteins involved in post-transcriptional regulation. These components will continue to grow, as future studies identify examples of direct communications between regulatory proteins, and reveal how gene networks are regulated co-ordinately through these interactions during stress in G. lamblia. The transcriptomic data has indicated towards some very important genes on which further studies can be carried out. They can be used as drug targets as this parasite also faces similar types of oxidative stresses inside our gut that has been mimicked here in vitro. The results have opened some new directions for further investigations. Protein characterization, signaling studies and further metabolomics can be carried out to achieve more interesting truths. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.11.048. Conflict of Interest There is no conflict of interest. Acknowledgment This study was jointly supported by a grant from the Okayama University Program of Founding Research Centre for Emerging and Reemerging Infectious Disease, Ministry of Education, Culture, Sports, Science and Technology of Japan, Govt. of Japan & the Indian Council of Medical Research, Govt. of India. The authors acknowledge Prof. S. Shinoda for his continuous constructive suggestions, support and critical review during this study. The authors also thank the University

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