Trichostatin A regulates peroxiredoxin expression and virulence of the parasite Entamoeba histolytica

Available online at www.sciencedirect.com

Molecular & Biochemical Parasitology 158 (2008) 82–94

Trichostatin A regulates peroxiredoxin expression and virulence of the parasite Entamoeba histolytica夽 Elada Isakov a , Rama Siman-Tov a , Christian Weber b,c , Nancy Guillen b,c , Serge Ankri a,∗ a

Department of Microbiology, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology and the Rappaport Institute, Haifa, Israel b Institut Pasteur, Unite de Biologie Cellulaire du Parasitisme, Paris F-75015, France c INSERM U786, Paris F-75015, France

Received 20 September 2007; received in revised form 4 November 2007; accepted 22 November 2007 Available online 4 December 2007

Abstract Histone deacetylation is associated with a repressed chromatin state, and histone acetylase and deacetylase activities have been previously described in Entamoeba histolytica. To investigate their roles in the control of Entamoeba gene expression, the parasite was grown in 50 nM trichostatin A (TSA), an inhibitor of histone deacetylase. TSA enhanced the cytopathic and hemolytic activity of the parasite and its resistance to oxidative stress. We first focused our attention on peroxiredoxin, a protein previously associated with E. histolytica virulence and resistance to oxidative stress. We found that the expression of peroxiredoxin was increased after TSA treatment, but were unable to confirm that this was a direct consequence of histone modification at the promoter. By microarray analysis, we found that some other mRNAs encoding some other virulence factors, such as the galactose-inhibitable lectin small subunits, were also increased. The pattern of gene expression was surprisingly different from that previously described after treatment with 150 nM TSA. © 2007 Elsevier B.V. All rights reserved. Keywords: Trichostatin; Entamoeba; Peroxiredoxin

1. Introduction The acetylation of the core histone N-terminal “tail” domains is now recognized as a highly conserved mechanism for regulating chromatin functional states. This mechanism is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation neutralizes the positive charge on histones, which disrupts higher-order structures in chromatin, thereby enhancing access of transcription factors, transcriptional regulatory complexes, and RNA polymerases to promoter regions of DNA. Histone deacetylation restores a positive charge on lysine residues of core histones, which allows chromatin to condense into a tightly supercoiled, transcriptionally silent conformation [1]. It is now well documented that 夽 Note: GenBank accession numbers are XM 643430, XP 648509, XM 642922, XM 649403, and XM 651789. ∗ Corresponding author at: Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O.B 9649, 31096 Haifa, Israel. Tel.: +972 4 829 5256; fax: +972 4 829 5225. E-mail address: [email protected] (S. Ankri).

0166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2007.11.014

aberrant transcription (i.e., epigenetic modulation) of genes that regulate cellular differentiation, the cell cycle and apoptosis is due to altered expression or mutation of genes that encode HATs, HDACs, or their binding and recruiting partners. Such modifications are key events in tumor onset and progression [2]. In protozoan parasites, the role of histone acetylation has recently been highlighted. In Giardia lamblia, the expression of variant-specific surface proteins (VSGs) seems to be dependent on histone acetylation [3]. In the malaria parasite Plasmodium, histone acetylation regulates gene expression during erythrocyte development [4]. For Toxoplasma gondii, histone acetylation is a marker of gene activation during differentiation, and promoter regions of tachyzoite-specific genes have been shown to be hyperacetylated in tachyzoites, but hypoacetylated during the bradyzoite stage [5]. In Trypanosoma brucei, the HDACs DAC1 and DAC3 are essential and DAC4 is required for cell cycle progression [6]. Indeed, T. brucei displays unique histone modifications such as acetylation of the multiple lysines of the C terminus of histone H2A (TbH2A) [7]. The components of the epigenetic machinery of Entamoeba histolytica are currently being studied. 5-Methylcytosine (m5C)

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

formation in repetitive DNA sequences and in heat shock protein 100 (Hsp100) is catalysed by Ehmeth, a protein that belongs to the DNA methyltransferase 2 family [8–11]. The presence of m5C in E. histolytica has recently been confirmed and extended to two virulence-related genes, cysteine proteinase and lysozyme [12]. We have previously shown that m5C in some specific repetitive sequences is sensed by a new protein called E. histolytica-methylated LINE binding protein EhMLBP [13]. The role of heterochromatin formation in the control of virulence gene expression in a number of pathogenic unicellular parasites has recently been reviewed [14]. Both HAT and HDAC activity has been detected in E. histolytica [15]. Interestingly, trichostatin A (TSA) [16] inhibits encystation of the reptile parasite Entamoeba invadens, which suggests that HDAC activity may have a role in parasite differentiation [17]. Recently, it has been reported that TSA (150 nM) induces the expression of encystation-related genes, such as the hypothetical protein 489.m00024 and heat shock proteins, that include Hsp70 [18], in E. histolytica strain 200:NIH. Indeed, several virulence-related genes, such as CP1 and the 35-kDa subunit of the Gal/GalNAc lectin are down-regulated by TSA in this strain [18]. Here, we report a quite different picture of the effect of TSA on gene expression in E. histolytica. TSA (50 nM) significantly enhanced in vitro virulence of HM-1:IMSS trophozoites. Peroxiredoxin and additional key genes participating in virulence, such as the light subunits of the Gal/GalNAc lectin, were identified among the genes that were up-regulated by TSA. The gene encoding Jacob, a protein involved in cyst formation, was also found to be activated by TSA. However, this phenotype did not fit with the regulation of other genes related to cyst formation, including stress-related proteins. The similarities and differences between the present study and that on strain 200:NIH [18] are discussed. This work emphasizes the role of epigenetic mechanisms in the control of E. histolytica virulence. 2. Materials and methods 2.1. Parasite and cell culture conditions Trophozoites of the E. histolytica strain HM1:IMSS were grown under axenic conditions in Diamond’s TYI-S-33 medium [19] at 37 ◦ C. Trophozoites in the log phase of growth were used in all experiments. HeLa cells were maintained in continuous culture in T75 tissue-culture flasks in Dulbecco’s modified Eagle’s medium (DMEM) (Beth HaEmek) supplemented with 4 mM l-glutamine, 1 mM sodium pyruvate, penicillin (100 U/ml), streptomycin (100 ␮g/ml) and 10% foetal calf serum. Cells were cultured at 37 ◦ C in a humidified 5% CO2 atmosphere.

83

− (DE3) (Stratagene), E. coli B F− dcm ompT hsdS (r− B mB ) gal l (DE3).

2.3. Previously published methodologies 2.3.1. Cytopathic activity The destruction rate of cultured HeLa cell monolayers by trophozoites grown with or without 50 nM TSA (Alomone Labs) for 48 h was determined as described previously [20,21]. Briefly, E. histolytica trophozoites were harvested by completely removing the existing medium (to remove cell debris, and dead and unbound trophozoites), adding fresh TYI-S-33 medium without serum, and chilling the tubes on ice. Following centrifugation at 700 × g for 10 min, the cell pellet was washed once with TYI-S-33 medium without serum, and viable trophozoites (5 × 105 ml−1 ) were incubated with the HeLa cell monolayers. 2.3.2. Adhesion assay Adhesion of trophozoites grown with or without 50 nM TSA for 48 h to HeLa cell monolayers was performed as previously described [21]. Trophozoites (2 × 105 ml−1 ) used in this study were harvested as described in Section 2.3.1. 2.3.3. Haemolytic activity The haemolytic activity of trophozoites grown with or without 50 nM TSA for 48 h was determined as previously described [22]. Trophozoites (2.5 × 105 ml−1 ) used in this study were harvested as described in Section 2.3.1, except for the last wash, which was carried out in PBS. 2.3.4. Hydrogen peroxide killing assay The resistance to oxidative stress induced by 2.5 mM hydrogen peroxide of trophozoites grown with or without 50 nM TSA for 48 h was determined as previously described [23]. Trophozoites were harvested as described in Section 2.3.1, and finally resuspended in DMEM supplemented with 70 mg l-cysteine and 135 mg ascorbic acid per 100 ml (DME-CH, pH 7.4) to a final concentration of 1 × 106 cells/ml. Trophozoites were incubated with 2.5 mM hydrogen peroxide for 1 h and their viability was determined by eosin exclusion. Transfection of E. histolytica trophozoites was performed as described in [11]. ChIP analysis was performed as previously described [13] using 5 ml of an antibody against pan-Acetyl (SANTA CRUZ). Preparation of nuclear acid-soluble proteins and acid/urea (AU) polyacrylamide gels were performed according to [24]. AU gels casted in a Mini-PROTEAN 3 cell (Bio-Rad) were used in this study. Nuclear-acid proteins where stained with the SilverQuest Silver staining kit (Invitrogen). This kit is compatible with mass spectrometry analysis.

2.2. Escherichia coli strains The phenotypes of the E. coli strains used in this study were as follows. XL1-Blue (Stratagene), recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F proAB lacIq ZDM15 Tn10 (tetr )]; BL-21

2.4. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Protein bands of interest were excised in gels digested with trypsin following a standard protocol [25] and analyzed by

84

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

MALDI-TOF MS (for a review see [26]), which was carried out at the Institute of Biology, Technion, Israel. The peptide-mass profiles produced by MALDI-TOF MS were analyzed using PepMiner (described at http://www.haifa.il.ibm/ projects/verification/bioinformatics/). Peptides masses were compared with the theoretical masses derived from the sequences contained in the SWISS-PROT/TrEMBL (http:// www.expasy.ch/sprot/), NCBI (http://www.ncbi.nml.nih.gov/ web/genbank) and the E. histolytica Genome Project databank (http://tigrblast.tigr.org/). 2.5. Northern blot analysis For Northern blot hybridization, total RNA was prepared using a TRI-reagent solution (Sigma). Total RNA (10 ␮g) was size-fractionated on a 4% polyacrylamide denaturing gel containing 8 M urea under denaturing conditions, and subsequently blotted electrophoretically onto a nylon membrane. Hybridization was carried out with DNA probes randomly labelled using the Random Primer DNA Labeling Mix Kit (Beit Haemeck). Membranes were washed after overnight hybridization using a non-stringent buffer (0.1% SDS, 2× SSC), followed by a stringent buffer (0.1% SDS, 0.1× SSC). Detection was carried out by autoradiography. 2.6. Construction of recombinant GST/E. histolytica peroxiredoxin Recombinant E. histolytica peroxiredoxin (accession number XM 643430) was prepared from the prokaryotic expression vector system pGEX-4T-1 (Pharmacia Biotech). This vector allows the expression of a protein fused to a GST tag. E. histolytica peroxiredoxin was amplified by PCR from genomic DNA with the primers GST-peroxiredoxin5 and GST-peroxiredoxin3 (Supplementary data S1). The PCR product was cloned in a pGEMT easy vector (Promega), digested with BamHI and NotI, and subcloned into the pGEX-4T-1 plasmid, which was previously linearized with BamHI and NotI (pGEX-4T-1Ehperoxiredoxin). 2.7. Expression of recombinant GST/E. histolytica peroxiredoxin For the expression of GST/E. histolytica peroxiredoxin, Escherichia coli BL-21 (DE3) transfected with pGEX-4T-1Ehperoxiredoxin was grown overnight in Luria Broth (LB) medium containing ampicillin (100 ␮g/ml). The preculture was inoculated (1:100) with 2× YT medium supplemented with ampicillin (100 ␮g/ml) and grown for ∼2 h at 37 ◦ C until OD600 reached 0.8. Induction of the fusion protein was initiated by adding isopropyl-␤-d-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to the growing culture. After 4 h incubation at 30 ◦ C, the bacteria were harvested and lysed in BugBuster Protein Extraction Reagent (Novagen). The recombinant GST/E. histolytica peroxiredoxin protein was purified under native conditions on gluthatione/agarose resin (Sigma). The recombinant proteins were then eluted with glutathione

elution buffer [Tris/HCl 50 mM, pH 8.0, glutathione (Sigma) 10 mM] and their concentrations were measured by Bradford’s method [27]. 2.8. Production of anti-peroxiredoxin antibody Male BALB/c and A/J mice were immunized intraperitoneally with 100 ␮g GST/E. histolytica peroxiredoxin recombinant protein emulsified in complete Freund’s adjuvant. Two and four weeks later, the mice were boosted with 100 ␮g recombinant protein in incomplete Freund’s adjuvant. One week after the final boost, ∼0.8 ml immune serum was obtained by retroorbital puncture. A non-immune serum was obtained from mice that had not been immunized. 2.9. Western blot analysis Proteins were separated on 10% polyacrylamide SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was exposed to Ponceau S (Sigma) to verify the efficiency of the transfer. Blots were then blocked (5% bovine serum albumin), and reacted with mouse polyclonal anti-E. histolytica peroxiredoxin antibody (1:750). After incubation with the first antibody, the blots were subjected to interaction with an HRP-conjugated goat anti-mouse antibody (1:10,000) (Jackson ImmunoResearch) and developed by enhanced chemiluminescence. The mouse anti-actin (monoclonal clone C:4) (1:7500) used in this study was from MB Biomedicals. The rabbit anti-pan-Acetyls (c8663-r) (1:500) used in this study was from SANTA CRUZ. 2.10. Detection of reactive oxygen species (ROS) production Control and stress-derived E. histolytica trophozoites were incubated with 0.4 mM (final concentration) 2 ,7 dichlorofluorescein diacetate (H2 DCFDA; Sigma) for 15 min in the dark. The cells were washed twice in PBS (pH 7.4) and immediately examined under a confocal microscope (LSM510; Zeiss). DCF fluorescence was also detected by a FACSCalibur flow cytometer (Becton Dickinson) equipped with a single laser system. Cells were excited with 488 nm light, and emission was measured through 530/30 (FL1). 2.11. Sodium bisulfite reaction and strand-specific PCR The EpiTect Bisulfite kit (Qiagen) was used to perform sodium bisulfite treatment of E. histolytica genomic DNA. The primers used to amplify peroxiredoxin (5 CHIP-peroxiredoxin and 3 CHIP-peroxiredoxin) after treatment with sodium bisulfite are described in Supplementary data S1. 2.12. Microarray gene expression profiling The oligonucleotide microarray is an updated version of the precedent chip [28] essentially carrying information from

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

transcript from parasites in vegetative growth and cytoskeletonrelated genes. These were enriched with probes that recognize transcripts from the following genes: cysteine proteinases, glycoproteins, DNA repair, gene lateral transfert, TM-Kinases, glycosyl transferases, BspA and heat shock factors; as well as genes encoding unknown proteins found in proteomic analysis [29]. In total, probes recognizing 2478 genes were spotted. Microarrays were hybridized, washed and dried as previously described [28]. Slides were scanned (GenePix 4000A scanner; Axon) and images were analyzed (GenePix5 software; Axon). The photomultiplier (PMT) gains were set to 640 (635 nm) and 550 (532 nm) for all slides. For each spot, the fluorescence value corresponded to the median pixel intensity within the spot (the GenePix F635 and F532 median columns). Two independent biological replicates were carried out in the experiment; two additional technical replicates were performed. Moreover, a dye swap was performed for each technical replicate to compensate biases introduced by the use of Cy3 and Cy5. Therefore, the experiment finally yielded eight hybridized slides. For normalization and differential analysis, scripts written with R software (http://www.R-project.org) were used. Lowess normalization was performed on all spots Entamoeba probes on a slide-by-slide basis (BioConductor marray package; http://www.bioconductor.org). After pooling data from the technical and biological replicates, a differential analysis was carried out. The raw P values were adjusted by the Benjamini and Yekutieli method, which controls the false discovery rate (FDR). We considered genes with a Benjamini and Yekutieli P value <0.05 and ≥2-fold change in expression as being differentially expressed. An expression threshold, i.e., the mean of empty spots intensity + 2 S.D., was calculated on each slide. All data sets are available in Supplementary Table S2, and detailed analyses are also available at http://genoscript.pasteur.fr. Click on public area, select Entamoeba, experiment TSA, and search for significant results. Data can be ordered by ratio, gene name, FDR or other displayed columns. 2.13. RT-PCR analysis Total RNA was prepared from trophozoites using a TRIreagent solution (Sigma). Reverse transcription was performed with the EZ-First Strand cDNA Synthesis Kit for RT-PCR (Biological Industries), according to the manufacturer’s instructions. Primers used to amplify E. histolytica Lgl2, Lgl3, Lgl5, senescence-related protein (SRP), chitinase, chitin synthase, Jacob and chloramphenicol acetyl transferase (CAT) are described in Supplementary data S1. Direct sequencing of the PCR product was performed to confirm the specificity of the reaction. Ethidium bromide fluorescence incorporated in Lgl2, Lgl3, Lgl5 and SRP PCR products following 20 cycles was analyzed and quantified using the TINA quantification software (raytest; Isotopenmessgerate, Straubenhardt, Germany). 2.14. Construction of pRCAT and pRCATPERO vectors The CAT gene linked to 3 actin was amplified from pEhNeoCAT (kindly given by Dr. E. Tannich, Hamburg, Germany) with

85

primers 3 actinApaI and 5 CATkpnI (Supplementary data S1) and cloned in the pGEM T easy vector (Promega) (pGEMCAT). The neomycin resistance gene was isolated from pEhActNEo [30] restricted with ApaI and KpnI, and cloned in pGEMCAT, which was previously linearized with ApaI and KpnI, to give the pRCAT vector. The peroxiredoxin putative promoter region was amplified with primers 5 prompero and 3 prompero (Supplementary data S1) and cloned in the KpnI site of pRCAT to give the pRCATPERO vector. 3. Results 3.1. Treatment of trophozoites with TSA inhibits their growth and affects the acetylation state of histone H4 Encystation of E. invadens was inhibited by TSA, and trophozoites exposed to this compound had increased levels of histone H4 and H2B acetylation [17]. We first determined the concentration of TSA that affected histone acetylation, but which maintained significant growth of E. histolytica. TSA (100 nM) strongly inhibited the growth of E. histolytica, which suggests that HDAC activity is essential for the parasite (Fig. 1A). TSA (50 nM) interferes fairly (40% inhibition) with the growth of the parasite exposed to the drug for 48 h (Fig. 1A). To determine the direct effect of TSA on E. histolytica HDAC activity, we measured its activity as previously described [15]. Unfortunately, we were unable to reproduce the measurement of this activity in different preparations of E. histolytica nuclear protein extracts, whereas HDAC activity in HeLa cell nuclear extracts was successfully measured with the HDAC assay kit (Upstate) (data not shown). This result suggests that the substrate provided in this HDAC assay kit is not appropriate for E. histolytica HDAC. The efficiency of 50 nM TSA to inhibit E. histolytica HDAC activity was therefore measured indirectly by the accumulation of acetylated histones. Acid-soluble nuclear proteins from control and E. histolytica trophozoites cultivated with 50 nM TSA were run in duplicate on one-dimensional acid/urea (AU) polyacrylamide gels. One gel was silver stained (Fig. 2) and the second was blotted onto a PVDF membrane. The blotting efficiency was confirmed by staining the membrane with Ponceau Red (data not shown). Commercial antibodies that cross-react with E. histolytica acetylated histones are unavailable [17]. For that reason, the membrane was incubated with a panacetyl lysine antibody (Fig. 2). This antibody recognizes an acetylated lysine independently of the amino acid sequence that flanks the modified lysine. A band is recognized by the pan-acetyl lysine antibody in control lysate. This band had a stronger intensity in the lysate prepared from trophozoites treated with 50 nM TSA. To determine the nature of this protein, a part of the gel that included this band was excised from the silver-stained AU gel, destained, digested with trypsin, and subjected to liquid chromatography–tandem mass spectrometry amino acid sequences analysis. The most abundant peptides (Supplementary Table S3) corresponded to E. histolytica histone H4 (accession number XP 648509).

86

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

Fig. 1. Growth, virulence and oxidative stress assays in trophozoites grown with TSA. (A) Dose–response effect of TSA on the growth of E. histolytica. Data represent the mean and standard deviation of three independent experiments. Diamond’s TYI-S-33 media (7 ml) that contain different concentration of TSA (0–100 nM) were inoculated with vegetative growing trophozoites (5 × 104 ) and incubated 48 h at 37 ◦ C. Trophozoites were harvested by directly chilling the tubes on ice, counted, and their viability was determined by their ability to exclude the vital dye eosin (0.1%). The number of viable trophozoites that were harvested from the cultures without TSA was taken as 100%. (B) Cytopathic activity was measured as the ability of amoeba grown in the presence of TSA (50 nM) for 48 h to destroy a monolayer of HeLa cells. The ± column shows the cytopathic activity of trophozoites grown for 48 h with TSA (50 nM) and then cultivated without TSA for 1 week. Data represent the mean and standard deviation of three independent experiments carried out in duplicate. Destruction of the monolayer was determined by incorporation of methylene blue dye into remaining cells, as previously described [20]. (C) Haemolytic assay measuring the ability of trophozoites grown in the presence of TSA (50 nM) for 48 h to lyse human red blood cells. The haemoglobin in the supernatant was read at 570 nm in a spectrophotometer. Data represent the mean and standard deviation of three independent experiments carried out in duplicate. (D) Oxidative stress assay measuring the ability of trophozoites grown in the presence of TSA (50 nM) for 48 h to survive exposure to hydrogen peroxide (2.5 mM) for 1 h (trophozoite survival = number of living trophozoites following hydrogen peroxide exposure/number of living trophozoites before hydrogen peroxide exposure). The viability of the exposed trophozoites was measured by their ability to exclude eosin. Data represent the mean and standard deviation of three independent experiments carried out in duplicate.

The presence of acetylated lysine in histone H4 of HM1:IMSS untreated trophozoites (Fig. 2) contradicted a recent study, which showed an absence of detectable lysine acetylation in histone H4 of this strain [31]. To validate our result, 40 ␮g acid-soluble nuclear proteins prepared from HM1:IMSS trophozoites were run on a 15% SDS polyacrylamide gel. Part of the gel that included proteins with a molecular mass of ∼15 kDa was subjected to liquid chromatography–tandem mass spectrometry amino acid sequences analysis. As shown in Supplementary Table S4, acetylation of lysine K5, K8, K12 and K15 in peptides that belong to E. histolytica histone H4 was successfully detected by this method. The presence of differentially acetylated peptides suggests that the band recognized by the panacetyl antibody (Fig. 2) represented a combination of different acetylated histone H4 species, rather than a single acetylated species. Taken together, these results indicate that 50 nM TSA is an adequate working concentration because it affects histone H4 acetylation and maintains significant growth of the parasite.

pathic and haemolytic activities (Fig. 1B and C) when compared with untreated parasites. Indeed, the resistance of the trophozoites to oxidative stress generated by hydrogen peroxide was also significantly enhanced in the TSA-treated cells (Fig. 1D). No difference was found between untreated and TSA-treated trophozoites for their adhesion to HeLa cell monolayers, or their erythrophagocytosis activity (data not shown). Epigenetic regulation is synonymous with reversible modification of gene expression. Therefore, we tested the virulence of trophozoites that were grown for 2 days with 50 nM TSA and then cultivated without TSA for 1 week. We observed that their cytopathic and haemolytic activities and resistance to 2.5 mM hydrogen peroxide recovered to wild-type levels equivalent to those of untreated trophozoites (Fig. 1B–D). These results show that the effect of TSA on E. histolytica virulence is reversible.

3.2. Treatment of trophozoites with TSA increased in vitro virulence and resistance to hydrogen peroxide

Expression of peroxiredoxin has been associated with increased resistance to oxidative stress and virulence in E. histolytica [32]. The phenotype of trophozoites treated with TSA led us to focus our analysis on the expression of peroxiredoxin. Northern hybridization was carried out in order to measure the

Trophozoites that were grown for 2 days in the presence of 50 nM TSA showed a significant increase in their cyto-

3.3. Expression of peroxiredoxin in TSA-treated trophozoites

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

87

Fig. 2. AU gel. Acid-soluble nuclear proteins were extracted from E. histolytica trophozoites grown in either TYI-S-33 medium or TYI-S-33 medium plus TSA (50 nM) and fractionated (2 ␮g/lane) in AU gels in duplicate. One gel was silver stained (left panel) and the second was blotted onto nitrocellulose membrane. Red Ponceau staining of the nitrocellulose membrane was used to control transfer efficiency (data not shown). The membrane was incubated with pan-acetyl lysine antibody (right panel). The same band intensity was observed for two independent lysates. The curly bracket shows a part of the gel that has been excised and subjected to MS. The molecular mass written close to the bracket corresponds to the theoretical molecular mass of E. histolytica histone H4.

transcription level of peroxiredoxin in trophozoites treated with TSA (Fig. 3A). We observed that transcripts encoding peroxiredoxin were more abundant in trophozoites grown with TSA compared to untreated trophozoites. The higher expression of peroxiredoxin genes in the presence of TSA was correlated with an increase in protein level, as assessed by Western blotting using a specific GST/peroxiredoxin antibody obtained in this study (Fig. 3B). Both the 30- and 60-kDa protein bands that represent the peroxiredoxin monomer and dimer, which are resistant to reducing conditions [23], were more intense in the lysates of trophozoites treated with TSA (Fig. 3B). Interestingly, the expression of peroxiredoxin returned to the control level in trophozoites that were grown for 2 days with 50 nM TSA and then cultivated without TSA for 1 week (Fig. 3B). A possible explanation for the induction of peroxiredoxin expression by TSA is that this drug induces directly or indirectly the formation of ROS. To test this hypothesis, we determined the level of ROS within living trophozoites using H2 DCFDA, which was converted into highly fluorescent DCF in the presence of ROS. Immunofluorescence microscopy showed very little fluorescence in control and TSA-treated trophozoites, whereas intense fluorescence was observed in hydrogen peroxide-exposed trophozoites (Fig. 4A). This result was confirmed by FACS analysis (Fig. 4B), which indicates that TSA does not induce the formation of ROS in trophozoites. TSA causes selective loss of DNA methylation in Neurospora [34] and we have shown that it induces DNA demethylation in E. histolytica Hsp100 [9]. Nevertheless, DNA methylation was not detected by bisulfite sequencing in the promoter region and

Fig. 3. Peroxiredoxin expression in trophozoites grown with TSA. (A) Northern blot analysis of peroxiredoxin. Total RNA (10 ␮g) was size-fractionated on 4% polyacrylamide denaturing gel containing 8 M urea under denaturing conditions, and subsequently blotted electrophoretically onto a nylon membrane. Hybridization was carried out by probes for the coding region of actin or peroxiredoxin. These probes were prepared from E. histolytica cDNA that was amplified with primers actin5 and actin3 for the actin probe, and with primers 5 CHIP-peroxiredoxin and 3 CHIP-peroxiredoxin for the peroxiredoxin probe (primers are described in Supplementary Table S1). (−) Control trophozoites; (+) trophozoites grown in the presence of TSA (50 nM). The data are representative of two independent experiments. (B) Western blot analysis, under reducing conditions, of trophozoite total lysate (25 ␮g) probed with anti-peroxiredoxin antibody (1/750) or anti-actin antibody (1/5000). (−) Control trophozoites; (+) trophozoites grown in the presence of TSA (50 nM); (±) trophozoites grown for 2 days with TSA (50 nM) and then cultivated without TSA for 1 week.

the open reading frame of peroxiredoxin (data not shown). This result rules out a possible role for DNA methylation in the control of peroxiredoxin expression by TSA. TSA can modulate gene expression by acting directly on histone acetylation, or indirectly by affecting transcription factor expression. For example, the activities of transcription factors Sp1 and Sp3, which control extracellular superoxide dismutase gene expression in mammals [35], and NF-␬B, which controls cyclin D1 expression [36], are affected by TSA. In an effort to understand how TSA regulates the expression of peroxiredoxin, we decided to clone the 5 -potential promoter region of the E. histolytica peroxiredoxin gene (from −538 bp upstream of the ATG). This region was amplified from genomic DNA

88

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

Fig. 4. Visualization of ROS production. (A) Control trophozoites, and trophozoites grown in the presence of TSA (50 nM) for 2 days, or exposed to hydrogen peroxide (2.5 mM for 1 h), were incubated with H2 DCFDA and examined by epifluorescence microscopy. Note that fluorescence only appeared when the parasites where treated with hydrogen peroxide. (B) DCF fluorescence was also detected by a FACSCalibur flow cytometer (Becton Dickinson) equipped with a single laser system. Cells were excited with 488 nm light, and emission was measured through 530/30 (FL1). An arbitrary gate has been applied to avoid taking into account cellular debris.

(primers are described in Supplementary Table S1) and inserted upstream of a CAT reporter gene carried by the pRCAT vector. The resultant vector was named pRCATPERO. Trophozoites stably transformed with the pRCATPERO vector were grown for 48 h with and without TSA, and the amount of CAT transcript was measured by RT-PCR. We observed no difference in the amount of CAT transcript between TSA-treated and untreated trophozoites (data not shown), which suggests that up-regulation of peroxiredoxin expression by TSA is not mediated by the activity of transcription factors potentially binding to DNA sequences upstream of the peroxidase-encoding gene. Chromatin immunoprecipitation (Chip) with antibodies raised against acetylated specific histones is a method currently used to determine histone modifications across the promoter and coding regions of genes of interest. Except for the methylated K4 of histone H3 [37], amoebic histone modifications are not recognized by commercially available histone-specific antibodies, due to a divergence in their amino acid sequence [17]. A panacetylated antibody that widely recognizes acetylated lysine was therefore used to perform the Chip analysis. With this rough tool, we were unable to detect a difference in the amount of peroxiredoxin gene precipitated out by the pan-acetylated antibody between trophozoites grown with and without TSA (data not shown). In addition, this inconclusive result emphasizes our need for specific antibodies against E. histolytica acetylated histones. 3.4. Microarray expression profiling reveals genes modulated by TSA treatment To elucidate the mechanism by which TSA enhances the virulence of E. histolytica, we carried out a cDNA microarray

analysis using an updated microarray, which carries E. histolytica oligonucleotides that are able to identify 2500 unique transcripts. The first conclusion was that peroxiredoxin genes were up-regulated in the presence of TSA. Nevertheless, the first computational analysis of the identified factors showed that peroxiredoxin-encoding genes were up-regulated (1.8–2 times) in one biological replicate, and very highly expressed in the second biological replicate (see Supplementary data S2) according to the saturating fluorescent signals for this gene. Overall, these data confirm the Northern blotting results, which indicated an overexpression of peroxiredoxin-encoding genes (Fig. 3A). To obtain an overview of our microarray results, we classified genes according to function (Fig. 5A): 15% of the up-regulated genes were related to virulence, compared to 3% of the down-regulated genes. In contrast, 21% of the down-regulated genes were related to cellular organization and biogenesis, compared to 6% of the up-regulated genes. Around 40% of the up- and down-regulated genes potentially encode proteins of unknown function. Due to the stringent statistical analysis of microarray data, we retained in the compiled list those genes that were modulated at least twofold in the presence of TSA. Thus, we found that 13 genes were significantly up-regulated by TSA (Table 1), while 31 genes were down-regulated (Table 2). One of the most striking findings of the microarray analysis was the differential expression of alleles for the light-chain subunit of the Gal/GalNac lectin, which is an abundant surface protein complex involved in contact-dependent killing and phagocytosis of target cells by E. histolytica [38]. We found that the Lgl-2 and Lgl-3 alleles, and to a lesser extent Lgl-1, were up-regulated by TSA, whereas all other light-chain subunits were not. This result was confirmed by semi-quantitative RT-PCR analysis (Fig. 5B).

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

89

Table 1 Genes up-regulated by TSA Oligo identity

NCBI accession no.a

Identification

Fold change

BY × VMb

EH-IP0334.2 EH-IP0035 EH-IP1917 EH-IP2197 EH-IP2001 EH-IP0816.2 EH-IP0816.1 EH-IP1380 EH-IP1244 EH-IP2135 EH-IP0869.1 EH-IP1941 EH-IP0144 EH-IP1341

202.t00007 101.t00003 33.t00050 6.t00023 4.t00058 56.t00038 56.t00038 142.t00016 110.t00009 520.t00001 628.t00001 35.t00042 131.t00014 133.t00014

20 kDa antigen-related protein Conserved hypothetical protein Cyst wall-specific glycoprotein Jacob Endo-1,4-beta-xylanase, putative Galactose-inhibitable lectin small subunit Lgl-2 Galactose-inhibitable lectin small subunit Lgl-3 Galactose-inhibitable lectin small subunit Lgl-3 Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Iron–sulfur flavoprotein Iron–sulfur flavoprotein Metal-dependent hydrolase

2.1 2.4 3.0 2.0 2.0 3.4 2.6 2.9 2.2 2.1 2.4 2.7 2.2 2.1

2.19E−03 1.85E−03 4.23E−05 4.09E−04 1.16E−02 2.16E−04 1.28E−03 6.70E−06 1.69E−06 4.70E−05 1.89E−06 1.16E−02 1.01E−03 1.28E−03

a b

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nuccore. p value according to [57].

Table 2 Genes down regulated by TSA Oligo identity

NCBI accession no.a

Identification

Inhibition in fold change

BY × VMb

EH-IP0461.2 EH-IP0461.1 EH-IP0826.2 EH-IP0970.4 EH-IP2284 EH-IP0111.2 EH-IP2505 EH-IP0095 EH-IP1212 EH-IP1580 EH-IP1154 EH-IP0036.1 EH-IP0055.1 EH-IP1234 EH-IP0218 EH-IP1658 EH-IP1760 EH-IP0628.4 EH-IP0628.2 EH-IP0708.1 EH-IP0708.2 EH-IP2120.1 EH-IP2120.3 EH-IP2136 EH-IP0807.4 EH-IP0821.2 EH-IP0821.6 EH-IP0927.2 EH-IP2386 EH-IP2405 EH-IP0415 EH-IP0764.1 EH-IP0764.3 EH-IP0764.2 EH-IP0375

27.t00041 27.t00041 59.t00014 88.t00015 71.t00021 12.t00062 348.t00004 118.t00013 103.t00038 119.t00006 101.t00009 101.t00009 107.t00005 11.t00046 160.t00017 218.t00002 26.t00025 36.t00042 36.t00042 43.t00031 43.t00031 50.t00024 50.t00024 523.t00007 54.t00042 58.t00022 58.t00022 79.t00022 90.t00032 96.t00013 248.t00012 5.t00037 5.t00037 5.t00037 22.t00027

Actin-like Actin-like Actin-binding protein Amoebapore A precursor Conserved hypothetical protein Conserved hypothetical protein Cortexillin II Cortexillin II Fe-hydrogenase Heat shock protein 70 Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Leucine-rich repeat protein Myosin heavy chain Myosin heavy chain Myosin heavy chain Villin-related protein

2.1 2 2.1 2.1 3 2 2.1 2 2.3 2.1 3.1 2.9 2.3 2.2 2.1 2.4 2.1 2.1 2 2 3.1 2.4 2.4 2.2 2.2 2.3 2 2.1 2.3 2.2 2.5 2.9 2.6 2.4 2.8

9.78E−05 2.26E−04 1.18E−02 6.58E−03 1.56E−05 6.58E−03 4.46E−03 1.22E−02 7.53E−05 1.67E−03 3.95E−06 1.16E−05 8.90E−03 2.97E−02 9.38E−04 5.46E−03 9.06E−04 7.52E−03 3.44E−03 1.66E−02 6.58E−05 5.21E−03 6.84E−03 1.90E−02 1.50E−02 2.92E−03 2.35E−02 7.17E−03 8.11E−03 1.49E−02 4.90E−04 2.26E−04 1.22E−03 2.07E−03 5.50E−04

a b

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nuccore. p value according to [57].

90

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

Fig. 5. (A) Categories of genes whose transcription is up- or down-regulated by TSA treatment. The data were organized according to a number of different categories suggested for the analysis of transcripts from protozoan parasites (http://www.ebi.ac.uk/parasites/ old/FalcipProteome/geneclass.html). Note that a homogeneous colour code (appearing in a clockwise representation) is associated with each category. The distribution of total genes was obtained by the percentages of different categories of genes carried on the microarray ([28] and this work). (B) Quantification of mRNA encoding the lectin light subunit by RT-PCR. Array data were confirmed for the differential expression of alleles for the light-chain subunit of the Gal/GalNac lectin by semi-quantitative RT PCR (left panel). The primers used for PCR amplification of Lgl2, Lgl3, Lgl5 and SRP [10] are described in Supplementary Table S1. Up-regulation of Lgl2 and Lgl3 in the presence of TSA was confirmed by RT-PCR. Lgl5, whose expression did not change (based on array data) in trophozoites grown with TSA, was found to be unchanged by RT-PCR. Values were normalized by taking the value of densitometric quantification for SRP.

Jacob is the most abundant glycoprotein of amoebic cyst walls [39]. This glycoprotein is specifically expressed by E. invadens during encystation and in response to heat shock [40]. We observed that the transcription of Jacob is increased roughly threefold in trophozoites cultivated with TSA (Table 1). The expression of two other cyst-specific genes, chitinase [41] and chitin synthase [42], was determined separately by semiquantitative RT-PCR, as these genes were not represented in the DNA array that was used in this study. RT-PCR confirmed that transcription of Jacob was increased by TSA (data not shown). However, we were unable to detect significant expression of chitinase and chitin synthase in the control and TSA-treated trophozoites (data not shown).

Impairment of E. histolytica cytoskeleton activity is associated with low phagocytosis activity and virulence [43]. The most down-regulated genes under treatment with TSA are involved in cellular organization and biogenesis, such as cortexillin and actin-related proteins (Table 2), which indicates that cytoskeleton-related activity, as yet undescribed, may be affected by TSA. One concern with the sub-lethal concentration of TSA (50 nM) used in this study was that the drug may have induced a stress response in the parasite. The results of our microarray analysis ruled out this concern. We showed that peroxiredoxin expression was up-regulated by TSA, whereas it was strongly inhibited following heat shock (7–19-fold) [28]. Indeed, stress-

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

91

Table 3 Genes regulated by TSA and 5-AzaC (according to [12]) NCBIa accession no.

5-AzaC (fold change)

TSA (fold change)

Identification

52.t00007 99.t00020 420.t00001 4.t00070 40.t00023 415.t00001 51.t00028 214.t00016 98.t00023 29.t00026 43.t00031 248.t00012 58.t00022 1.t0001 59.t00022 100.t00006 458.t00007 134.t00007 2.t00055 4.t00061 6.t00023 520.t00001 35.t00042

0.55 2.4 0.34 2.9 0.66 0.56 0.57 3.6 2.6 0.68 2.2 1.8 2.4 1.7 0.53 0.59 4.2 0.54 0.27 0.33 1.66 3.2 0.32

0.565 0.573 0.576 0.58 0.59 0.61 0.63 0.64 0.7 0.73 0.32 0.4 0.54 0.66 1.48 1.5 1.7 1.87 1.88 1.94 2.05 2.1 2.7

Lysoyme putative Hypothetical protein Pre-rRNA processing protein rrp5 Hypothetical protein Hypothetical protein Cortexillin Protein kinase Elongation factor 2 Pyruvate:ferredoxin oxidoreductase Calcium binding protein Hypothetical protein Leucine-rich repeat protein Hypothetical protein Actin-like protein TPR repeat protein Hypothetical protein Hypothetical protein HSP20 Cysteine proteinase EF-hand calcium binding protein Endo-1,4-beta-xylanase Hypothetical protein Iron–sulfur flavoprotein

In bold are represented genes that are inversely regulated by TSA and 5-AzaC. All genes modulated by TSA (even under statistical scores) have been taken into account. a http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nuccore.

related proteins like the 90-kDa Hsp70, DNAJ and EhSSP1 were strongly induced in heat-shocked trophozoites [28] and following oxidative stress for Hsp70 and EhSSP1 [44,45], whereas none of these proteins was significantly up-regulated by TSA. A microarray analysis of the effect of TSA (150 nM) on gene expression in E. histolytica 200:NIH has recently been carried out [18]. We compared the profiles of gene expression reported in Ehrenkaufer et al. [18] and our study (strain HM-1:IMSS; 50 nM TSA). Only one gene (hypothetical protein; accession number XM 642922) was commonly up-regulated by TSA and two genes (lysozyme, putative; accession number XM 649403 and Beige BEACH domain protein, putative; accession number XM 651789) were down-regulated by TSA. DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. Therefore, it was interesting to compare the expression of genes following treatment with 5-azacytidine (5-AzaC) to inhibit DNA methylation [12], and treatment with TSA. We observed that 14 out of 23 genes sensitive to 5-AzaC and TSA were inversely modulated by these drugs (Table 3), and only a few genes such as the lysozyme gene were co-regulated. These results suggest that DNA methylation and histone deacetylation control different sets of genes in E. histolytica. 4. Discussion TSA has been widely used to study the role of histone acetylation in gene expression and is considered a promising antitumor agent [45]. TSA has been shown to inhibit encystation of the reptile parasite E. invadens [17] and the growth of E. histolyt-

ica ([18] and this work). The present study demonstrated that the virulent strain of E. histolytica HM-1:MSS grown in the presence of TSA (50 nM) showed a significant increase in virulence compared to that in untreated trophozoites. Three main classes of virulence factors have been extensively characterized in recent years in E. histolytica: the Gal/GalNAc-specific lectin [46], cysteine proteinases [47,48], and the amoebapores [49]. The virulent phenotype observed in trophozoites grown with TSA and their remarkable resistance to oxidative stress may be, in part, related to the up-regulation of peroxiredoxin expression. The mechanism linked to histone deacetylase enhancing transcription of peroxiredoxin-encoding genes and synthesis of this protein in the presence of TSA is unknown. We did not find any differences in ROS, DNA methylation, activity of potential transcription factors, or chromatin epigenetic modification. However, peroxiredoxin is a surface molecule that protects the parasite from host-generated oxidant attack [23,33]. This protein is also involved in metronidazole resistance [50] and it is molecularly associated with the Gal/GalNAc lectin [51]. Interestingly, peroxiredoxin has a significantly lower expression in the non-pathogenic Entamoeba dispar than in E. histolytica HM1:IMSS [32], and it has recently been demonstrated that this protein is an important component of amoebic virulence [52]. In oesophageal cancer cells, peroxiredoxin expression is turned off by histone H3 and H4 deacetylation in the promoter region of this gene, and can be reactivated by HDAC inhibitors [53]. It will be interesting, once specific antibodies against acetylated amoebic histones become available, to analyse the acetylation status of these histones in the promoter region of E. histolytica peroxiredoxin.

92

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

It seems unlikely that changes in the expression of a single, or even a few gene products can explain the phenotypes observed in trophozoites cultivated with TSA. Therefore, DNA microarray analysis allowed us to perform a wide analysis of additional genes that are regulated by histone acetylation. Among the genes up-regulated by TSA, the galactose-inhibitable lectin small subunits are of particular interest. The correlation between their expression and parasite virulence has previously been established [21]. The Gal/GalNAc lectin is composed of two subunits, a light (Lgl), glycosylphosphatidylinositol-anchored 31/35-kDa subunit (Lgl) and a heavy 170-kDa subunit (Hgl). The complex is associated with an intermediate 120-kDa subunit (Igl). Each lectin subunit is represented in the E. histolytica genome by multiple alleles; however, the functional distinctions between these alleles have not yet been established [38]. Previous studies have shown that the light lectin subunits expression is modulated by environmental factors such as heat shock [28] and bacterial flora [54]. Interestingly, the effect of bacterial flora on the expression of light Gal/GalNAc lectins is reversible [54]. It is remarkable that, in different organisms, genes regulated by stress or environmental factors are also regulated by an epigenetic mechanism. This is illustrated in rice, in which submergence-inducible genes are regulated by reversible changes in histone H3/K4 methylation and H3 acetylation [55], or in mammals, in which oxidative stress induces the expression of pro-inflammatory genes, following changes in the acetylation pattern of nuclear histones [56]. In E. histolytica, our previous work has shown that Hsp100 expression is regulated by both thermic stress and TSA [9]. Hsp100 was not carried by the DNA array used in this study, which explains its absence from the list of genes up-regulated by TSA. An interesting example is represented by Jacob, the most abundant glycoprotein of amoebic cyst walls [39]. This glycoprotein is specifically expressed by E. invadens during encystation and in response to heat shock [40]. Recently, expression of this protein has been described in encysting E. histolytica trophozoites. We observed that Jacob was expressed specifically in trophozoites treated with TSA, although no additional markers of encystation, such as chitinase and chitin synthase, were expressed. Data from the DNA array analysis of trophozoites grown with TSA indicated that these trophozoites were not subjected to harsh stress, as the expression level of Hsps was rather low or non-existent and TSA did not generate oxidative stress. Therefore, the expression of Jacob seems to be epigenetically regulated; however, the significance of this regulation for the differentiation of the parasite needs to be studied separately. A microarray analysis that describes the effect of TSA on gene expression in E. histolytica has recently been published [18]. A very different gene expression profile was observed between that and the present study, with only three genes that were commonly regulated. A major difference with our study was the strong induction by TSA of stress-related genes. A second difference was the down-regulation of several virulencerelated genes, including the 35-kDa Gal/Gal-NAc lectin that was up-regulated in our study. These differences may be partially explained by the choice of strain (HM-1:IMSS versus 200:NIH) and the concentration of TSA that was used (50 nM

versus 150 nM TSA). Another possibility is that the two studies were simply looking at two different physiological responses to TSA. As mentioned by the authors of the previous study, a stress response to TSA cannot be ruled out [28]. In summary, we showed that TSA modulated the virulence of E. histolytica HM-1:IMSS. Different phenotypes and expression profiles observed in strain 200:NIH treated with TSA emphasize the complexity of this parasite. The present study, together with recently published works on this subject, contributes to a better understanding of the epigenetic machinery present in this parasite. Acknowledgements We thank Jean Yves Copp´ee and Odile Sismeiro (Genopole– Pasteur Institute) for their help in the setting up of microarray experiments, Gishlaine Guigon for microarray statistical analysis, and Tamar Ziv (The Smoler Protein Center-Technion) for her help with the mass spectrometry analysis. This study was supported by grants from the Israel Science Foundation, Center for the Study of Emerging Diseases, Israeli Ministry of Health, the Rappaport Institute and the Pasteur–Weizmann Research Council. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2007.11.014. References [1] Shahbazian MD, Grunstein M. Functions of site-specific histone acetylation and deacetylation. Annu Rev Biochem 2007;76:75–100. [2] Dey P. Chromatin remodeling, cancer and chemotherapy. Curr Med Chem 2006;13:2909–19. [3] Kulakova L, Singer SM, Conrad J, Nash TE. Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation. Mol Microbiol 2006;61:1533–42. [4] Miao J, Fan Q, Cui L, Li J. The malaria parasite Plasmodium falciparum histones: organization, expression, and acetylation. Gene 2006;369:53– 65. [5] Saksouk N, Bhatti MM, Kieffer S, et al. Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii. Mol Cell Biol 2005;25:10301–14. [6] Ingram AK, Horn D. Histone deacetylases in Trypanosoma brucei: two are essential and another is required for normal cell cycle progression. Mol Microbiol 2002;45:89–97. [7] Janzen CJ, Fernandez JP, Deng H, Diaz R, Hake SB, Cross GA. Unusual histone modifications in Trypanosoma brucei. FEBS Lett 2006;580:2306–10. [8] Banerjee S, Fisher O, Lohia A, Ankri S. Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR). Mol Biochem Parasitol 2005;139:91–7. [9] Bernes S, Siman-Tov R, Ankri S. Epigenetic and classical activation of Entamoeba histolytica heat shock protein 100 (EHsp100) expression. FEBS Lett 2005;579:6395–402. [10] Fisher O, Siman-Tov R, Ankri S. Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica. Nucleic Acids Res 2004;32:287–97. [11] Fisher O, Siman-Tov R, Ankri S. Pleiotropic phenotype in Entamoeba histolytica overexpressing DNA methyltransferase (Ehmeth). Mol Biochem Parasitol 2006;147:48–54.

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94 [12] Ali IK, Ehrenkaufer GM, Hackney JA, Singh U. Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression. BMC Genomics 2007;8:7. [13] Lavi T, Isakov E, Harony H, Fisher O, Siman-Tov R, Ankri S. Sensing DNA methylation in the protozoan parasite Entamoeba histolytica. Mol Microbiol 2006;62:1373–86. [14] Sullivan Jr WJ, Naguleswaran A, Angel SO. Histones and histone modifications in protozoan parasites. Cell Microbiol 2006;8:1850–61. [15] Ramakrishnan G, Gilchrist CA, Musa H, et al. Histone acetyltransferases and deacetylase in Entamoeba histolytica. Mol Biochem Parasitol 2004;138:205–16. [16] Yoshida M, Kijima M, Akita M, Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem 1990;265:17174–9. [17] Byers J, Faigle W, Eichinger D. Colonic short-chain fatty acids inhibit encystation of Entamoeba invadens. Cell Microbiol 2005;7:269– 79. [18] Ehrenkaufer GM, Eichinger DJ, Singh U, Trichostatin. A effect on gene expression in the protozoan parasite Entamoeba histolytica. BMC Genomics 2007;8:216. [19] Diamond LS, Harlow DR, Cunnick CC. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 1978;72:431–2. [20] Bracha R, Mirelman D. Virulence of Entamoeba histolytica trophozoites. Effects of bacteria, microaerobic conditions, and metronidazole. J Exp Med 1984;160:353–68. [21] Ankri S, Padilla-Vaca F, Stolarsky T, Koole L, Katz U, Mirelman D. Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence. Mol Microbiol 1999;33:327–37. [22] Ankri S, Stolarsky T, Mirelman D. Antisense inhibition of expression of cysteine proteinases does not affect Entamoeba histolytica cytopathic or haemolytic activity but inhibits phagocytosis. Mol Microbiol 1998;28:777–85. [23] Choi MH, Sajed D, Poole L, et al. An unusual surface peroxiredoxin protects invasive Entamoeba histolytica from oxidant attack. Mol Biochem Parasitol 2005;143:80–9. [24] Shechter D, Dormann HL, Allis CD, Hake SB. Extraction, purification and analysis of histones. Nat Protoc 2007;2:1445–57. [25] Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996;68: 850–8. [26] Mann M, Hendrickson RC, Pandey A. Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem 2001;70:437–73. [27] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [28] Weber C, Guigon G, Bouchier C, et al. Stress by heat shock induces massive down regulation of genes and allows differential allelic expression of the Gal/GalNAc lectin in Entamoeba histolytica. Eukaryot Cell 2006;5: 871–5. [29] Marion S, Laurent C, Guillen N. Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach. Cell Microbiol 2005;7: 1504–18. [30] Alon RN, Bracha R, Mirelman D. Inhibition of expression of the lysine-rich 30 kDa surface antigen of Entamoeba dispar by the transcription of its antisense RNA. Mol Biochem Parasitol 1997;90:193– 201. [31] Byers J, Eichinger D. Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner. Int J Parasitol 2008;38(1):57–64. [32] MacFarlane RC, Singh U. Identification of differentially expressed genes in virulent and nonvirulent Entamoeba species: potential implications for amebic pathogenesis. Infect Immun 2006;74:340–51. [33] Poole LB, Chae HZ, Flores BM, Reed SL, Rhee SG, Torian BE. Peroxidase activity of a TSA-like antioxidant protein from a pathogenic amoeba. Free Radic Biol Med 1997;23:955–9.

93

[34] Selker EU, Trichostatin. A causes selective loss of DNA methylation in Neurospora. Proc Natl Acad Sci USA 1998;95:9430–5. [35] Zelko IN, Folz RJ. Sp1 and Sp3 transcription factors mediate trichostatin A-induced and basal expression of extracellular superoxide dismutase. Free Radic Biol Med 2004;37:1256–71. [36] Hu J, Colburn NH. Histone deacetylase inhibition down-regulates cyclin D1 transcription by inhibiting nuclear factor-kappaB/p65 DNA binding. Mol Cancer Res 2005;3:100–9. [37] Anbar M, Bracha R, Nuchamowitz Y, Li Y, Florentin A, Mirelman D. Involvement of a short interspersed element in epigenetic transcriptional silencing of the amoebapore gene in Entamoeba histolytica. Eukaryot Cell 2005;4:1775–84. [38] Petri Jr WA, Haque R, Mann BJ. The bittersweet interface of parasite and host: lectin–carbohydrate interactions during human invasion by the parasite Entamoeba histolytica. Annu Rev Microbiol 2002;56:39– 64. [39] Frisardi M, Ghosh SK, Field J, et al. The most abundant glycoprotein of amebic cyst walls (Jacob) is a lectin with five Cys-rich, chitin-binding domains. Infect Immun 2000;68:4217–24. [40] Field J, Van Dellen K, Ghosh SK, Samuelson J. Responses of Entamoeba invadens to heat shock and encystation are related. J Eukaryot Microbiol 2000;47:511–4. [41] de la Vega H, Specht CA, Semino CE, et al. Cloning and expression of chitinases of Entamoebae. Mol Biochem Parasitol 1997;85: 139–47. [42] Campos-Gongora E, Ebert F, Willhoeft U, Said-Fernandez S, Tannich E. Characterization of chitin synthases from Entamoeba. Protist 2004;155:323–30. [43] Marion S, Wilhelm C, Voigt H, Bacri JC, Guillen N. Overexpression of myosin IB in living Entamoeba histolytica enhances cytoplasm viscosity and reduces phagocytosis. J Cell Sci 2004;117:3271–9. [44] Akbar MA, Chatterjee NS, Sen P, et al. Genes induced by a highoxygen environment in Entamoeba histolytica. Mol Biochem Parasitol 2004;133:187–96. [45] Vanhaecke T, Papeleu P, Elaut G, Rogiers V. Trichostatin A-like hydroxamate histone deacetylase inhibitors as therapeutic agents: toxicological point of view. Curr Med Chem 2004;11:1629–43. [46] Gilchrist CA, Petri WA, Singh VK, Moskovitz J, Wilkinson BJ, Jayaswal RK. Virulence factors of Entamoeba histolytica. Curr Opin Microbiol 1999;2:433–7. [47] Que X, Reed SL. Cysteine proteinases and the pathogenesis of amebiasis. Clin Microbiol Rev 2000;13:196–206. [48] Bruchhaus I, Loftus BJ, Hall N, Tannich E. The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes, of which only a small subset is expressed during in vitro cultivation. Eukaryot Cell 2003;2:501–9. [49] Leippe M, Herbst R. Ancient weapons for attack and defense: the poreforming polypeptides of pathogenic enteric and free-living amoeboid protozoa. J Eukaryot Microbiol 2004;51:516–21. [50] Wassmann C, Hellberg A, Tannich E, Bruchhaus I. Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase. J Biol Chem 1999;274:26051–6. [51] Hughes MA, Lee CW, Holm CF, et al. Identification of Entamoeba histolytica thiol-specific antioxidant as a GalNAc lectin-associated protein. Mol Biochem Parasitol 2003;127:113–20. [52] Davis PH, Zhang X, Guo J, Townsend RR, Stanley Jr SL. Comparative proteomic analysis of two Entamoeba histolytica strains with different virulence phenotypes identifies peroxiredoxin as an important component of amoebic virulence. Mol Microbiol 2006;61:1523– 32. [53] Hoshino I, Matsubara H, Hanari N, et al. Histone deacetylase inhibitor FK228 activates tumor suppressor Prdx1 with apoptosis induction in esophageal cancer cells. Clin Cancer Res 2005;11:7945–52. [54] Padilla-Vaca F, Ankri S, Bracha R, Koole LA, Mirelman D. Down regulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the

94

E. Isakov et al. / Molecular & Biochemical Parasitology 158 (2008) 82–94

light (35-kilodalton) subunit of the Gal/GalNAc lectin. Infect Immun 1999;67:2096–102. [55] Tsuji H, Saika H, Tsutsumi N, Hirai A, Nakazono M. Dynamic and reversible changes in histone H3-Lys4 methylation and H3 acetylation occurring at submergence-inducible genes in rice. Plant Cell Physiol 2006;47:995–1003.

[56] Rahman I. Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J Biochem Mol Biol 2003;36:95–109. [57] Reiner A, Yekutieli D, Benjamini Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 2003;19:368–75.