Comparison of hemolytic activity of the intermediate subunit of Entamoeba histolytica and Entamoeba dispar lectins

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica has roles in pathogenicity and induction of protective immunity in rodent models of amoebiasis. Recently, the intermediate subunit of the lectin, Igl1, of E. histolytica has been shown to have hemolytic activity. However, the corresponding lectin is also expressed in a non-virulent species, Entamoeba dispar, and another subunit, Igl2, is expressed in the protozoa. Therefore, in this study, we compared the activities of Igl1 and Igl2 subunits from E. histolytica and E. dispar using various regions of recombinant Igl proteins expressed in Escherichia coli. The recombinant E. dispar Igl proteins had comparable hemolytic activities with those of E. histolytica Igl proteins. Furthermore, Igl1 gene-silenced E. histolytica trophozoites showed less hemolytic activity compared with vector-transfected trophozoites, indicating that the expression level of Igl1 protein influences the activity. These results suggest that the lower hemolytic activity in E. dispar compared with E. histolytica reflects the lower expression level of Igl1 in the E. dispar parasite.


Introduction
Amoebiasis due to infection with Entamoeba histolytica (E. histolytica) is a problematic parasitic disease in many countries. E. histolytica causes an estimated 50 million cases of dysentery, colitis and extraintestinal abscesses, resulting in 40,000 to 100,000 deaths annually [1]. Adherence of E. histolytica trophozoites to colonic mucins and host cells is mediated by a galactose (Gal)-and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin [2]. This lectin is a 260-kDa heterodimer of two glycoproteins: a transmembrane heavy subunit (Hgl, 170 kDa), one of the key molecules in amebic adherence, and a glycosylphosphatidylinositol (GPI)-anchored light subunit (Lgl, 35/31 kDa).
Another GPI-anchored 150-kDa intermediate subunit (Igl, 150 kDa) is non-covalently associated with the Hgl/Lgl dimer in different lipid raft-like domains and also contributes to adherence [3][4][5]. There are two isoforms of Igl, which are referred to as Igl1 and Igl2, and both contain multiple CXXC motifs with different localization in E. histolytica trophozoites [6,7]. These two Igls are also found in Entamoeba dispar (E. dispar), which is morphologically indistinguishable from E. histolytica, but is non-pathogenic [8]. The expression level of Igl1 is about twice as high in E. histolytica HM-1:IMSS than in E. dispar SAW1734RclAR, whereas that of Igl2 is comparable in the two species, suggesting that Igl1 may be associated with the pathogenicity of E. histolytica [8]. In fact, Igl1 is recognized by sera from patients with amoebiasis and is also a vaccine candidate [9,10]. Igl is a parasitic lectin that binds to p-aminophenyl-β-D-galactopyranoside-Sepharose gel in a Gal-affinity column [5]. E. histolytica Igl has also been detected, in addition to Hgl and Lgl, in the protein fraction that binds to GalNAc bovine serum albumin-coated magnetic beads [11]. Recently, while exploring the lectin domain of Igl, we found that Igl1 of E. histolytica possesses both hemolytic and cytotoxic activities [12]. However, it is unclear whether Igl1 of E. dispar and Igl2 of both species have the same activity. Therefore, in this study, we compared the hemolytic activities of E. dispar Igls with those of E. histolytica Igls in vitro. We also attenuated expression of Igl1 in E. histolytica utilizing a gene-silencing technique and evaluated the effect on hemolytic activity, since E. dispar has lower expression of Igl1 compared with E. histolytica [8].

Materials and methods
Expression and refolding of recombinant Igl proteins and Ni column purification of the proteins Recombinant EhF-Igls, EdF-Igls, EhNM-Igl1, EhM-Igl1, EhC-Igl1 or EdC-Igl1 proteins with a His-tag at the N-terminus were expressed in Escherichia coli BL21 Star(DE3)pLysS cells (Invitrogen) or ECOS™ competent BL21(DE3) cells (Nippon Gene Co.), using the primers shown in Table 1. The proteins were further purified using a Ni column, as described in detail previously [9,12]. Table 1. Oligonucleotide primers used in the study.

SDS-PAGE and coomassie brilliant blue staining of purified recombinant proteins
Recombinant proteins (1 μg each) were mixed with SDS sample buffer (Invitrogen) and subjected to SDS-PAGE. The gel was treated with SimplyBlue Safe stain solution (Invitrogen) and incubated until blue bands appeared on the gel [12].
Hemolytic assays using recombinant lectins and measurement of released hemoglobin Hemolytic assays and quantification of hemolytic activity were conducted as previously described [12]. Briefly, recombinant Igls (2 μM each, 50 μl) were mixed with 50 μl of horse red blood cell (HoRBC) solution at room temperature and images were taken at several time points. A Hemoglobin B Test Kit (Wako, Osaka, Japan) was used to measure the concentration of hemoglobin in supernatants of RBCs incubated with recombinant proteins or trophozoites for 8 h or 1 h. The results are expressed as the mean of 5 experiments with SD.

Real-time PCR analysis
Real-time PCR was essentially performed as previously described [8]. Briefly, total RNAs of E. histolytica and E. dispar trophozoites isolated using a RNeasy Plus Mini Kit (Qiagen) were used for cDNA synthesis with an ExScript™ RT Reagent Kit (Takara). Reaction mixtures for quantitative real-time PCR analysis were prepared using SYBR Premix Ex Taq II (Takara), specific primers, Rox dye, and the cDNAs. Forty cycles of amplification with recording of fluorescence intensity in each cycle were performed using StepOnePlus™ Real-Time PCR System (ABI). Expression levels of Igl genes were analyzed using the comparative C T method with actin as an internal standard. The experiments were repeated 3 times, including the steps of culture and isolation of RNA.

Antibodies
Purified human mAb XEhI-20 (anti-EhIgl1) and XEhI-B5 (anti-EhIgl2) were used for detection of Igls of E. histolytica [7]. Mouse mAb ED2-495 against recombinant Igl2 of E. dispar was obtained as described previously [8] and characterized before use. Mouse mAb ED2-1 specific for Igl2 of E. dispar was used as a control [8]. Pooled ascites rich in mouse mAbs were used in the study.

Dot blot analysis
Recombinant Igls (300 ng) were blotted on a nitrocellulose membrane and then air-dried. Filter strips were blocked with 3% bovine serum albumin in PBS and reacted with ED2-1 or ED2-495 for 30 min. After washing with PBS containing 0.05% Tween-20 (PBST), the strips were incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (MP Biomedicals) for 30 min. The strips were then washed with PBST and developed with a Konica Immunostaining HRP-1000 kit.

Immunoblot assay
Whole cell lysates (15 or 20 μg protein/well) were applied to a 5-20% gradient polyacrylamide gel (Atto Corp., Tokyo, Japan) and SDS-PAGE was conducted under reducing or non-reducing conditions, respectively. The proteins in the gel were transferred onto an Amersham™ Hybond™ P 0.45 PVDF membrane (GE Healthcare) that was then incubated with 5% skim milk in PBST for blocking. Mouse ascites (ED2-495) against E. histolytica Igls was diluted 500 times with PBST containing 5% skim milk. Rabbit antiserum against ATP sulfurylase (AS) of E. histolytica was prepared [16] and diluted 500-fold with 5% skim milk in PBST. Anti-mouse and anti-rabbit immunoglobulin F(ab')2 fragments conjugated with HRP (Amersham) were diluted 3000 times with PBST and used as the secondary antibody. Immunoblot assays with XEhI-20 or XEhI-B5 [7] were performed using SDS-PAGE in a 5-20% gradient gel under non-reducing conditions, with 30 μg of XEhI-20 or XEhI-B5 used as the primary antibody and HRP-labeled goat anti-human IgG antibody (MP Biomedicals) diluted 1000 times with PBST as the secondary antibody. Immobilon™ Western (Millipore) was used as a substrate for visualization of the proteins. Detection of chemiluminescence and quantification of band intensities were performed by Ez-Capture MG and CS Analyzer ver. 3.0 (Atto Corp.), respectively.

Immunofluorescence assay
Sample preparation for IFA was performed as previously described [18,19]. Briefly, after amoeba transformants were incubated on 5-mm round wells on glass slides, the cells were fixed with 4% paraformaldehyde in PBS for 10 min, washed four times with PBS, and permeabilized with 0.05% Triton X-100 in PBS for 5 min. After blocking with 3% bovine serum albumin in PBS, samples were reacted with ED2-495 (mouse IgG), XEhI-20 (human IgG), or XEhI-B5 (human IgG) diluted 1:50 in PBS and subsequently reacted with secondary antibody diluted 1:500 (Alexa Fluor 1 488 goat anti-mouse or human IgG; Life Technologies) in PBS. Fluorescence images were obtained using a LSM510 Meta confocal Microscope (Zeiss) in lambda emission fingerprinting mode [20,21].

Hemolytic assay using Entamoeba histolytica and Entamoeba dispar trophozoites
The assay was conducted as previously described [12,22] with slight modifications. Briefly, vector-transfected (control) or Igl1 gene-silenced (gsIgl1A or gsIgl1B) E. histolytica or wild-type E. dispar (SAW1734RclAR strain or CYNO9:TPC strain) trophozoites prepared as described above were harvested and washed with PBS. Then 1×10 5 trophozoites were mixed with 1% HoRBCs in 100 μl PBS and incubated at 37˚C for 1 h. The cell suspension was sedimented at 2000 rpm for 5 min and the concentration of hemoglobin in the supernatant was determined as described above.

Statistical analysis
Multiple comparisons were performed by ANOVA with a Dunn test, with P < 0.05 considered significant.  (Fig 1) were expressed in E. coli [12]. Recombinant proteins were purified using Ni columns, the buffer was changed to PBST, and purities were confirmed by SDS-PAGE (Figs  2A and 3A). The recombinant proteins were then used in further studies.

Hemolytic activities of Entamoeba dispar strains
We have reported that E. histolytica trophozoites have hemolytic activity and that the activity can be blocked by an antibody recognizing M/C-Igl of EhIgl1 [12]. To assess whether E. dispar trophozoites also have this activity, the trophozoites were incubated with HoRBCs for 1 hr. The trophozoites of E. dispar SAW1734RclAR strain were cultured with Pseudomonas aeruginosa or with Crithidia fasciculata monoxenically because they were difficult to culture axenically. Trophozoites of another E. dispar strain, CYNO9:TPC, were able to be cultured axenically and the hemolytic activity of this strain was also assessed. As shown in Fig 4, SAW1734RclAR trophozoites cultured with Pseudomonas aeruginosa had hemolytic activity (Fig 4A and 4B) while trophozoites of the same strain cultured with Crithidia fasciculata and CYNO9:TPC strain did not have activity under the test conditions. The activity in SAW1734R-clAR trophozoites cultured with Pseudomonas aeruginosa is due to contamination of hemolysin expressed in Pseudomonas aeruginosa [23].

Hemolytic activity of Igl1 gene-silenced Entamoeba histolytica
Recombinant Igl proteins from E. histolytica and E. dispar showed similar hemolytic activities. However, expression of Igl1 in trophozoites of E. histolytica is about twice as high as that in E. dispar trophozoites [8]. At the same time, E. dispar trophozoites did not have hemolytic activity (Fig 4). To evaluate whether the expression level of Igl1 affects hemolytic activity, we generated Igl1 gene-silenced (gsIgl1) E. histolytica strains and conducted the hemolytic assay.  Comparison of lectin activities of Entamoeba species Expression levels of Igl1 and Igl2 in the gene-silenced E. histolytica trophozoites were evaluated quantitatively by real-time PCR (Fig 5). E. dispar trophozoites expressed about a half the level of Igl1 compared with E. histolytica trophozoites (Cont), as described previously [8]. Both Igl1 gene-silenced E. histolytica strains (gsIgl1A and gsIgl1B) showed significantly lower expression of Igl1, but not Igl2, compared with vector control trophozoites (Fig 5). We could not generate an Igl2 gene-silenced E. histolytica strain for unknown reason. Interestingly, Igl2 expressions in Igl1-silenced E. histolytica strains were rather high compared with that in vector control trophozoites (Fig 5).
Downregulation of Igl1 was further confirmed by Western blotting and cell staining (Fig 6). Western blotting by mAb ED2-495, which recognizes both Igl1 and Igl2 of E. histolytica and E. dispar (Fig 6A), showed 50-80% reduction of Igl expression in gsIgl1A and gsIgl1B strains, with this reduction due mainly to downregulation of Igl1 (Fig 6B and 6C). Expression levels of Igl2 in gsIgl1A and gsIgl1B strains were higher than in vector control trophozoites, in agreement with the real-time PCR results shown in Fig 5. Downregulation of Igl1 in gsIgl1 strains was also confirmed using an immunofluorescence assay (Fig 6D). These results indicate that establishment of the gsIgl1 strains was successful. After incubation of vector control or gsIgl1A trophozoites with HoRBCs for 1 h, the rim of the accumulated samples became vague in treatment with control trophozoites compared with gsIgl1A-treated samples (Fig 7A). For quantitative evaluation, supernatants of the incubated samples were collected and assayed for released Hb (Fig 7B). The hemolytic activity of gsIgl1A trophozoites (0.024±0.0066 g dL -1 ) was significantly lower than that of control trophozoites (0.060±0.0093 g dL -1 ). PBST treatment gave a [Hb] of 0.012±0.0065 g dL -1 . E. histolytica gsIgl1B trophozoites also had a lower hemolytic activity than control trophozoites (Fig 7C and  7D), indicating that lower hemolytic activity reflects lower Igl1 expression in the parasites.

Discussion
The E. histolytica lectin consists of three subunits, Hgl, Lgl and Igl, of which Hgl and Igl have lectin activities [24]. Recently, we found that EhIgl1 had hemolytic and cytotoxic activities [12]. Since E. dispar, a non-virulent species, also has an Igl1 subunit homologue, it is of interest to determine whether EdIgl1 has these activities. In this study, we showed that EdIgl1 has similar hemolytic activity to that of EhIgl1, with the site of this activity residing at the C-terminus in both proteins. There are two isoforms of Igl, and therefore, we also evaluated the hemolytic activities of EhIgl2 and EdIgl2. All EhIgls and EdIgls had hemolytic activity in our assay. This is the first study to show that E. dispar Igls and E. histolytica Igl2 lectins have hemolytic activities.
Factors related to the virulence of Entamoeba spp. remain unclear, despite several detailed studies [25][26][27][28]. Among the potential factors, both Hgl and Lgl lectin subunit expression are lower in E. dispar compared with E. histolytica [29]. Low expression of Lgl1 was also found in an avirulent E. histolytica Rahman strain compared with the highly virulent E. histolytica HM-1:IMSS strain [30]. Expression of dominant negative Hgl or Lgl in the E. histolytica HM-1: IMSS strain and antisense inhibition of expression of Lgl in the same strain gave a less virulent strain [30][31][32]. Antisense inhibition of expression of EhCP5, an amoebic cysteine protease, in Comparison of lectin activities of Entamoeba species the HM-1: IMSS strain resulted in reduced virulence [33][34][35]. EhCP5 is missing in E. dispar [36] and is expressed at a lower level in the Rahman strain compared to the HM-1:IMSS strain [25]. Antisense inhibition of amoebapore expression in the HM-1:IMSS strain also decreases amoebic virulence [37]. Thus, many virulence-related molecules have been identified, but there may be additional factors related to amoebic virulence [38].
The Igl subunit also has vital roles in the pathogenicity of the parasite, including attachment to host cells and killing activities [12,39,40]. EhF-Igl1 and EhF-Igl2 have 83-84% amino acid sequence identity, while the amino acid sequence identity of EhF-Igl1 and EdF-Igl1 is 75-76% and that of EhF-Igl2 and EdF-Igl2 is 73-74% [8]. EhC-Igl1 and EdC-Igl1 have 76-77% amino acid sequence identity, with conserved cysteine residues. The Igls also have different Comparison of lectin activities of Entamoeba species expression levels in each species, with Igl1 having higher expression in E. histolytica than in E. dispar, but Igl2 having similar expression in the two species [8]. To assess whether the difference in level of Igl1 proteins affects the hemolytic activity of E. histolytica, we generated Igl1 gene-silenced E. histolytica trophozoites and compared the activity with vector-transfected E. histolytica trophozoites. Interestingly, a 40% reduction of Igl1 protein expression led to a significant decrease in hemolytic activity.
This observation correlates with the weaker cytotoxicity of non-virulent E. dispar in a Gal/ GalNAc lectin-mediated manner in vitro [41]. Erythrophagocytosis of E. dispar was observed in another study [42]. More importantly, E. dispar is pathogenic in experimental animals [43][44][45][46] and in humans [47,48]. These effects have recently been reviewed [49]. The mechanism of the pathogenicity to E. dispar is still unclear, but it is possible that the incidence of infection by Entamoeba spp. is related to the expression levels of Igls and other related proteins. Further studies are needed to evaluate this possibility.
One of the interesting observations in this study was increased expression of Igl2 in both of Igl1-silenced strains. By contrast, decreased level of Igl2 expression has been observed by short hairpin RNA-mediated knockdown of Igl1 [50]. The discrepancy may be due to the difference of methods used to prepare transfectants.