GSNO was purchased from Sigma-Aldrich, St. Louis, MO, USA. CytD was purchased from Cayman Chemical Company, Ann Arbor, Michigan, USA.

E. histolytica trophozoites strain HM-1:IMSS were grown under axenic conditions in Diamond's TYI S-33 medium at 37°C. Trophozoites in the exponential phase of growth were used in all experiments. Trophozoites adapted to GSNO (120 μM) were prepared using a previously described protocol (Shahi et al., 2016b). Trophozoites exposed to an acute NS (TEANS) (1.5 10⁵ trophozoites/ml) were incubated for 60 min in Diamond's TYI S-33 medium at 37°C amended with 350 μM GSNO (final concentration). Trophozoites treated with CytD (1.5 10⁵ trophozoites/ml) were incubated for 12 h in Diamond's TYI S-33 medium at 37°C amended with 5 μM CytD (final concentration).

A total protein extract was prepared by lysing NAT (5 × 10⁷) in 1% Igepal CA-630 (Sigma-Aldrich, St. louis, Mo, USA) in phosphate buffer saline (PBS). S-nitrosylated proteins in the total protein extract were detected by SNO-RAC using a previously described protocol (Hertz et al., 2014a). Captured proteins were eluted with buffer (10 mM HEPES, 0.1 mM EDTA, 0.01 mM neocuproine, 0.1% SDS, 100 mM 2-mercaptoethanol) for 20 min at room temperature (RT), and the proteins in each eluent were resolved on a 12.5% SDS-PAGE gel. Each gel was then stained with Coomassie blue dye (Brilliant Blue G, Sigma-Aldrich, St. louis, Mo, USA) and each lane was submitted independently for mass spectrometric (MS) analysis.

For the detection of actin in SNO-proteins, an aliquot (10%) of the eluted proteins that were captured in absence or presence of ascorbate were resolved on a 10% SDS-PAGE in SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Proteins were electrotransferred in protein transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) to nitrocellulose membranes (Protran® BA83, Whatman). The blots were first blocked using 3% skim milk, and then probed with 1:1,000 monoclonal actin antibody (clone C4, MP Biomedicals, Solon, Ohio, USA) for 1 h at room temperature. The blots were incubated with 1:5,000 horseradish peroxidase conjugated secondary antibody (Jackson ImmunoResearch, Enco Diagnostics, Israel) for 1 h at RT, and then developed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific, USA).

The proteins in each gel slice were reduced with 2.8 mM DTT (60°C for 30 min), modified with 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate (room temperature for 30 min in the dark), and digested overnight in 10% acetonitrile and 10 mM ammonium bicarbonate with modified trypsin (Promega, Biological industries, Israel) at 37°C.

The resulting tryptic peptides were resolved by reverse-phase chromatography on 0.075 × 200-mm fused silica capillaries (J & W Scientific, Folsom, CA, USA) packed with ReproSil-Pur reversed phase material (Dr. Maisch GmbH, Germany). The peptides were eluted with a linear 95-min gradient of 7–40% and 8 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.25 μl/min. MS was performed by an ion-trap mass spectrometer (Orbitrap, Thermo Fisher Scientific, USA) in a positive mode of operation using a repetitively full MS scan followed by collision-induced dissociation (CID) of the seven most dominant ions selected from the first MS scan. The MS data was analyzed using Proteome Discoverer software (version 1.3) which searches the Ameba section of the NCBI non-redundant database and the decoy databases [in order to determine the false discovery rate (FDR)] using the Sequest and the Mascot search engines.

The online PANTHER Version 11.0 (http://pantherdb.org/; Rist et al., 2007) was used in this study. SNO proteins in NAT were classified by using the “protein class” ontology setting, the pie chart option and the percent of gene hit against total # Protein Class hits setting. The statistical overrepresentation test was performed using the default setting, the annotation data set corresponding to PANTHER protein class and the Bonferroni correction for multiple testing options selected.

Trophozoite motility was determined using the Costar Transwell system (8-μm pore size polycarbonate membrane, 6.5-mm diameter, Corning Inc, Corning, NY, USA; Gilchrist et al., 2008). For this purpose, trophozoites were first washed three times in serum-free Diamond's TYI-S-33 medium, and then suspended in serum-free Diamond's TYI-S-33 medium. A 500-μl aliquot of the suspension (25 × 10⁴ trophozoites/ml) was loaded into a transwell insert, which was placed in each well of a 24-well culture plate which contained serum-free Diamond's TYI-S-33 medium (500 μl/well). The 24-well culture plate with the loaded inserts was placed in anaerobic bags (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan), and incubated for 3 h at 37°C. At the end of the incubation, the inserts and culture medium were removed from each well, and trophozoite migration was determined by counting the number of trophozoites that were attached to the bottom of each well.

Erythrophagocytosis was assayed using a previously described protocol (Mora-Galindo et al., 1997). Briefly, Human red blood cells (Hrbcs) (5 × 10⁷) and trophozoites (5 × 10⁵) were mixed and incubated for 10 min at 37°C. Phagocytosis was stopped by adding distilled water. The average number of erythrocytes inside the trophozoites was quantified using a calibration curve and by reading the absorbance at 397 nm after suspending a pellet of parasites in 90% formic acid.

The rate of destruction of cultured HeLa cell monolayers by trophozoites was determined using a previously described protocol (Bracha and Mirelman, 1984). Briefly, E. histolytica trophozoites (2.5 × 10⁵ or 10⁵/well) in serum-free Diamond's TYI-S-33 medium were incubated with HeLa cell monolayers in 24-well tissue culture plates at 37°C for 60 min. The incubation was stopped by placing the plates on ice and unattached trophozoites were removed by washing the plates with cold PBS. The HeLa cells that remained attached to the plates were stained with methylene blue (0.1% in 0.1 M borate buffer, pH 8.7). The dye was extracted from the stained cells by 0.1 M HCl, and color intensity of extracted dye was measured spectrophotometrically at OD₆₆₀.

SUnSET was performed using a previously described protocol (Hertz et al., 2014b; Shahi et al., 2016a). Briefly, trophozoites (2 × 10⁶/ml) were incubated with 10 μg/ml puromycin (Sigma-Aldrich, St. louis, Mo, USA), a structural analog of tyrosyltRNA, for 20 min at 37°C. The trophozoites were lysed using 1% Igepal (Sigma) in PBS. Whole proteins were resolved on a 10% SDS-PAGE in SDS-PAGE running buffer. Proteins were electrotransferred in protein transfer buffer to nitrocellulose membranes. Loading equivalency was determined by immunoblotting using a 1:10,000 monoclonal α-tubulin antibody (DM1A clone, Sigma-Aldrich, St. louis, Mo, USA). Puromycin was detected by immunoblotting using a 1:5,000 monoclonal puromycin antibody (12D10 clone, Millipore). After incubation with the primary antibody, the blots were incubated with 1:5,000 secondary antibody for 1 h at RT (Jackson ImmunoResearch, Enco Diagnostics, Israel), and then developed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific, USA). Protein quantification/synthesis was estimated from the intensity of the immunoreactive blots (densitometry) using Fiji software (Schindelin et al., 2012).

E. histolytica trophozoites (1.5 10⁵ trophozoites/ml) were suspended in complete Diamond's TYI S-33 medium at 37°C and transferred onto acetone-cleaned glass coverslips that were placed in the bottom of each well of a 24-well plate. Trophozoites were incubated for 15 min at 37°C in order to allow them to adhere to the coverslip surface. The attached trophozoites were fixed with pre-warmed (37°C) 3.7% paraformaldehyde (PFA) for 30 min at RT. After fixation, the attached trophozoites were permeabilized with 0.1% Triton X-100/PBS for 1 min at RT. The coverslips were washed three times with PBS and quenched with PBS containing 50 mM NH₄Cl for 30 min at RT. The coverslips were then blocked with 1% bovine serum albumin (BSA) in PBS (BSA/PBS) for 30 min at RT. The samples were then probed with 1:500 monoclonal actin antibody (clone C4, MP Biomedicals, Solon, Ohio, USA) overnight. This monoclonal actin antibody was successfully used to detect E. histolytica actin (Perdomo et al., 2013) The next day, the samples were first washed three times in PBS, followed by two washes in 1% BSA/PBS, and then incubated with 1:250 Alexa Flour 488 (Jackson ImmunoResearch, PA, USA) and 1:1,000 4′,6-diamidino-2-phenylindole (DAPI) (MP Biomedicals, Solon, Ohio, USA) for 3 h at 4°C. At the end of the incubation, coverslips were incubated overnight at 4°C with 20 μM phalloidin [1 μM] conjugated to rhodamine (phalloidin conjugated to rhodamine was generously given by Prof. Adi Salzberg, Rappaport institute of Medicine, Technion, Haifa, Israel).

After incubation, the coverslips were washed three times in 1% BSA/PBS, and then with PBS. The samples were then mounted onto microscope slides with Fluoromount G (SouthernBiotech, Birmingham, AL, USA). The specimens were then examined under a confocal immunofluorescence microscope (ZEISS- LSM510 Meta Laser Scanning System confocal imaging system) with a 63X oil immersion objective.

Fluorescent quantification of F-actin in control trophozoites, NAT, NAT that have been cultivated for 1 month in absence of GSNO (NATR) and trophozoites treated with CytD was performed using Fiji software (Schindelin et al., 2012).

Data are presented as the mean ± standard deviation (SD). Significant differences between two groups were determined using an unpaired Student's t-test with a significance level of 0.05.

The amounts of SNO proteins in the untreated and ascorbate-treated (40 mM) total protein extract of NAT was determined by SNO-RAC coupled to MS (Hertz et al., 2014a; Figure 1A). A protein was considered to be a SNO protein when this protein was present in the ascorbate-treated lysates of at least two independent assays and not in the untreated lysates. From the results of three independent analyses, we found that 242 proteins fulfilled this criterion (Supplementary Table 1 and for more details Supplementary Tables 2, 3). These proteins included cytoskeletal protein (such as actin (EHI_159150) or paxillin (EHI_050720) (Table 1), signal transduction (such as GTP binding protein (EHI_148270) or RNA GTPase (EHI_148190), hydrolase (such as plasma membrane calcium transporting ATPase (EHI_016480), ligase [such as AcetylCoA synthase (EHI_135740) or long chain fatty acid-CoA ligase (EHI_153720)], Nucleic acid binding (this category is mostly represented by ribosomal proteins (RP) like 60 RPL2 (EHI_183480) or 40S RPS3a (EHI_065270), oxidoreductase (such as dihydropyrimidine dehydrogenase DPD (EHI_012980) or alcohol dehydrogenase (ADH) (EHI_198760) and transferase such as S-adenosyl-methionine synthetase (EHI_004920) and sulfate adenylyltransferase (EHI_197160) (Figure 1B). In order to confirm the consistency of our SNO-RAC analysis, actin was selected and the presence of SNO actin was confirmed by western blotting (Figure 1C). We observed that the amounts of actin which bound to the thiopropyl sepharose beads were significantly smaller in the untreated samples than the amounts in the ascorbate-treated samples. This result indicates that the binding of actin to the thiopropyl sepharose beads was due to its S-nitrosylation and not due to the background binding of the protein to the beads (Figure 1C). The results of the MS analysis of actin that was bound to the thiopropyl sepharose resin in the presence of sodium ascorbate revealed the presence of carbamidomethylated cysteine residues at positions 18, 218, and 286 (Supplementary Table 1). These residues possibly correspond to S-nitrosylated cysteines that have been reduced by the ascorbate, bound to the resin, eluted by 2- mercaptoethanol, and alkylated by iodoacetamide prior to digestion of the protein and MS analysis.

According to the results of the PANTHER statistical overrepresentation test, actin and actin related proteins, such as actin (EHI_159150) or orphan actin related protein (ARPvii; EHI_008780; fold enrichment 13 and P-value 3.53E⁻⁰⁵) and oxidoreductase, such as DPD (EHI_012980) or ADH (EHI_198760; fold enrichment 5.2 and P-value 1.53E⁻⁰³), were significantly enriched.

We found 27 common SNO proteins in NAT and in TEANS (Hertz et al., 2014a). According to the results of PANTHER statistical overrepresentation test, those with oxidoreductase activity and structural constituent of ribosome are the most enriched among these 27 SNO proteins (Table 2).

Only seven genes have both their expression differentially regulated in NAT (Shahi et al., 2016b) and have their product nitrosylated. These genes are plasma membrane calcium-transporting ATPase, (EHI_016480), 26s protease regulatory subunit (EHI_052050), phosphorylase (EHI_096830), myosin heavy chain (EHI_110180), helicase (EHI_148930), uncharacterized protein (EHI_189410), and a putative D-3-phosphoglycerate dehydrogenase (EHI_060860). According to the results of the PANTHER statistical overrepresentation test, the actin family of cytoskeletal proteins are significantly enriched among the products of upregulated genes in NAT (Shahi et al., 2016b) and SNO proteins in NAT (this work).

We previously reported that an acute NS inhibits protein synthesis in E. histolytica (Hertz et al., 2014a). The presence of proteins which are involved in translation among the SNO proteins in NAT, such as 60 RPL2 and 40S RPS3a, suggests that NO regulates the translation of proteins in NAT. In order to test this hypothesis, we used the SUnSET (Schmidt et al., 2009), to determine the amount of puromycin that was incorporated into nascent peptide chains (Figure 2). As previously described (Hertz et al., 2014b), we found that protein synthesis is strongly inhibited in TEANS compared to that in control (untreated) trophozoites. In contrast, we found that protein synthesis in NAT is comparable to that in control trophozoites (Figure 2).

The enrichment of actin family of cytoskeletal proteins among SNO proteins and among the products of genes that are upregulated in NAT (Shahi et al., 2016b) motivated us to investigate the presence of F-actin in control trophozoites and NAT by immunofluorescence microscopy with phalloidin, a molecule which selectively binds to F-actin. We found that the intensity of the F-actin signal in NAT is one third of that in control trophozoites (Figures 3A,B). In contrast, we previously found that the intensity of the NAOD signal or the GAPDH signal in NAT is the same of that in control trophozoites (Shahi et al., 2016b). NAOD or GAPDH are not included among SNO-proteins in NAT (Supplementary Table 1). CytD inhibits actin polymerization by binding to F-actin (May et al., 1998). We found that the same amount of F-actin was found in CytD-treated control trophozoites and NAT (Figures 3A,B). The finding of a reduced amount of F-actin in NAT and CytD-treated trophozoites was confirmed by immunofluorescence microscopy with an actin antibody (Figures 3A,B). Attempts to study the amount of F-actin in CytD-treated NAT were unsuccessful due to the high toxicity of CytD to NAT (data not shown). These results strongly suggest that GSNO impairs the formation of F-actin in NAT. Actin polymerization is essential for the phagocytic and cytopathic activities of E. histolytica (Godbold and Mann, 1998). A comparison between control trophozoites and NAT was carried out using the erythrophagocytosis assay (Figure 4A). The extent of erythrophagocytosis by NAT was half of that of the control trophozoites. We also found that erythrophagocytosis was impaired in CytD-treated control trophozoites (Figure 4A). Cytopathic activity of NAT was less than that of control trophozoites. Furthermore, CytD impairs the cytopathic activity of control trophozoites (Figure 4B). Since actin polymerization is an essential process during migration of the parasite (Emmanuel et al., 2015), we compared the migration of control trophozoites and NAT using the transwell migration assay. We found that the number of control trophozoites that passed through the pores was considerably greater than the number of NAT (Figure 4C). Furthermore, treatment of control trophozoites with CytD impairs their migration (Figure 4C). Collectively, these results indicate that virulence markers that depend on F-actin formation are impaired in NAT.

The fact that S-nitrosylation is a redox-reversible posttranslational protein modification (Gould et al., 2013) motivated us to determine if NAT phenotypes are reversible.

We previously reported that adaptation of E. histolytica to NO has a negative effect on its generation time (Shahi et al., 2016b). The generation time of NAT was half of that of control trophozoites (20 ± 1 h vs. 10.2 ± 0.5 h; p < 0.005). We also found that the generation time of NATR and control trophozoites are similar (10.3 ± 0.5 h). NAT can be continuously cultivated in presence of 110 μM GSNO (Shahi et al., 2016b). In contrast, NATR, like control trophozoites, died within 48 h when exposed to GSNO (120 μM; data not shown). Erythrophagocytosis, cytopathic activity, motility and the level of F-actin of NATR were tested. We found that the virulence and the level of F-actin in NATR and control trophozoites are similar (Figures 3A,B, 4A–C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.