INTRODUCTION
It has been estimated that 480 million people worldwide are infected with Entamoeba histolytica or Entamoeba dispar (Walsh, Reference Walsh1986). E. histolytica is the causative agent of human amoebic colitis and liver abscess, which result in up to 110 000 deaths annually. E. dispar is morphologically indistinguishable from E. histolytica, but is non-pathogenic and non-virulent (Diamond and Clark, Reference Diamond and Clark1993). Adherence of E. histolytica trophozoites to host cells is an essential step in its pathogenicity, and it is well known that the 170 kDa heavy subunit of galactose- and N-acetyl-d-galactosamine (Gal/GalNAc)-inhibitable lectin (Hgl) is the key factor in adherence and subsequent pathogenesis of the amoeba (Petri et al. Reference Petri, Haque and Mann2002). Hgl is a transmembrane protein that forms a heterodimer with a glycosylphosphatidylinositol (GPI)-anchored 35 kDa light subunit (Lgl) via disulfide bonds (Petri et al. Reference Petri, Chapman, Snodgrass, Mann, Broman and Ravdin1989). Recently, we have identified a GPI-anchored 150 kDa intermediate subunit (Igl) of lectin, which is non-covalently associated with Hgl (Cheng et al. Reference Cheng, Tsukamoto, Kaneda and Tachibana1998, Reference Cheng, Hughes, Huston, Loftus, Gilchrist, Lockhart, Ghosh, Miller-Sims, Mann, Petri and Tachibana2001). There are 2 isoforms of Igl, which consist of 1101 and 1105 amino acids and are referred to as Igl1 and Igl2, respectively; both are cysteine-rich proteins containing multiple CXXC motifs. A mouse monoclonal antibody (mAb) to Igl significantly inhibits adherence and cytotoxicity of trophozoites to mammalian cells in vitro and also inhibits liver abscess formation in hamsters (Tachibana et al. Reference Tachibana, Kobayashi, Cheng and Hiwatashi1997; Cheng et al. Reference Cheng, Kaneda and Tachibana1997, Reference Cheng, Tachibana and Kaneda1999). Antibodies to Igl have been detected not only in symptomatic patients with amoebiasis but also in asymptomatic cyst passers of E. histolytica (Tachibana et al. Reference Tachibana, Cheng, Masuda, Horiki and Takeuchi2004). Immunization of hamsters with affinity-purified Igl can prevent amoebic liver abscess formation (Cheng and Tachibana, Reference Cheng and Tachibana2001), and Igl has also been detected in the E. histolytica fraction that interacts with the brush border of enterocytes (Seigneur et al. Reference Seigneur, Mounier, Prevost and Guillen2005). Therefore, Igl seems to be one of the key molecules in amoebic adherence to host cells and pathogenicity; however, the correlation of these effects with each Igl isoform and the differences between the isoforms are not known.
Comparison of E. histolytica and E. dispar is also of importance for understanding the pathogenicity of amoeba. One well-known difference between the two species is associated with the family of cysteine proteases that are pathogenic factors in E. histolytica; in E. dispar, the EhCP1 gene is absent and EhCP5 is degenerate (Bruchhaus et al. Reference Bruchhaus, Jacobs, Leippe and Tannich1996; Willhoeft et al. Reference Willhoeft, Hamann and Tannich1999). Concerning lectins, it has been reported that Hgl is present in E. dispar, but that its expression level is lower than that in E. histolytica (Pillai et al. Reference Pillai, Britten, Ackers, Ravdin and Kain1997, Reference Pillai, Kobayashi and Kain2001). Therefore, it is of interest to determine if Igl1 and Igl2 are expressed in E. dispar. We report here the primary structure of the 2 Igl isoforms in 2 E. dispar strains isolated from human and cynomolgus monkey, respectively. We also compared the expression levels of Igl genes between E. dispar and E. histolytica, and examined the expression and localization of Igl1 and Igl2 in E. dispar.
MATERIALS AND METHODS
Cultivation of parasites
Trophozoites of the E. dispar SAW1734RclAR strain were grown axenically or monoxenically with sterilized Crithidia fasciculata in YIGADHA-S medium supplemented with 15% adult bovine serum at 37°C (Kobayashi et al. Reference Kobayashi, Imai, Haghighi, Khalifa, Tachibana and Takeuchi2005; Khalifa et al. Reference Khalifa, Imai, Kobayashi, Haghighi, Hayakawa and Takeuchi2006). Trophozoites of the E. dispar CYNO9:TPC strain were axenically cultured in the YIGADHA-S medium. Trophozoites of E. histolytica HM-1:IMSS were axenically cultured in TYI-S-33 medium supplemented with 15% adult bovine serum at 37°C (Diamond et al. Reference Diamond, Harlow and Cunnick1978). Cultured trophozoites were harvested in the logarithmic phase of growth and used in subsequent experiments.
Construction of a cDNA library and cloning of the Igl gene
Poly(A) RNA of E. dispar SAW1734RclAR trophozoites was isolated using a QuickPrep mRNA purification kit (Amersham Pharmacia). A cDNA library was constructed from 5 μg of poly(A) RNA using a cDNA synthesis kit (Amersham Pharmacia) and a λgt11 vector kit (Stratagene). The library was screened with a 657 bp probe using the Gene Images AlkPhos Direct labelling and detection system (Amersham Pharmacia). The probe was prepared from plasmid DNA containing the E. histolytica Igl1 gene by PCR amplification using primers EhIgl1-S877 (5′-CCC TCG AGT CAA ATG GTG AAT GTA AGC C-3′) and EhIgl1-AS1088 (5′-CCC TCG AGT TAA ATG CCT TTA GCT CCA TT-3′) (Tachibana et al. Reference Tachibana, Cheng, Masuda, Horiki and Takeuchi2004). The positive clone containing the longest insert was subcloned into a pUC19 vector and sequenced. To extend the sequence of the 5′ end, rapid amplification of the cDNA end was performed with a 5′-Full RACE Core Set (Takara). For the cloning of the other Igl gene, the cDNA library was subjected to PCR using primers 5′-CAA TTT CAC TTG GTG AGT ACA AAG CTG-3′ (forward) and 5′-GAA AAT TCC TTT ACT TCC ATT GCA GTT TCC-3′ (reverse). These primers were prepared based on the sequence of the first cloned E. dispar Igl gene, with reference to the location of common sequences between the two E. histolytica Igl genes. The amplified genes were cloned using a TOPO TA Cloning Kit (Invitrogen) and sequenced. To extend the sequence of the 5′ and 3′ ends of the cloned DNA, a 5′-Full RACE Core Set and 3′-Full RACE Core Set were used (Takara). For the cloning of Igl genes from the CYNO9:TPC strain, genomic DNA isolated as previously described (Tachibana et al. Reference Tachibana, Ihara, Kobayashi, Kaneda, Takeuchi and Watanabe1991) was used as a template for PCR, using the forward primers 5′-ATG TTT ATT ATT CTT TTA TTC ATA TCA ATT TCA C-3′ (Igl1) and 5′-ATG TTT ATT CTT CTT TTA TTT ATA TCA ATT TCA C-3′ (Igl2), and the reverse primer 5′-TTA GAA CAT AAA TGA TAA CAT GAC TAT CAC CAT C-3′. Thirty-five cycles of PCR using Pyrobest DNA polymerase (Takara) were performed as follows: denaturation at 94°C for 15 s (195 s in cycle 1), annealing at 58°C for 30 s, and polymerization at 72°C for 180 s (600 s in cycle 35). Amplified DNA was cloned using a Zero Blunt TOPO PCR Cloning Kit (Invitrogen) and sequenced. Nucleotide sequence data were analysed using Genetyx-Mac ver. 11.
Southern blot analysis
Genomic DNA was isolated from E. dispar SAW1734RclAR trophozoites as described previously (Tachibana et al. Reference Tachibana, Ihara, Kobayashi, Kaneda, Takeuchi and Watanabe1991). Three μg of genomic DNA was digested with restriction enzymes DraI, TaqI and HindIII. The fragments were separated on a 1% agarose gel, transferred to a Hybond N+ membrane (Amersham Pharmacia) by capillary action, and fixed by alkaline denaturation. The membrane was hybridized at 55°C in buffer containing a Gene Images AlkPhos Direct-labelled probe (Amersham Pharmacia) prepared by PCR amplification of cloned cDNA. The primers used for amplification were 5′-AGA TGG ATT CTA TTT TGA TGA-3′ (forward) and 5′-CAT ATG TCT TGA ACA TGG-3′ (reverse). The blots were detected using a CDP-star detection reagent (Amersham Pharmacia) and exposed to autoradiography films.
Real-time RT-PCR analysis
Total RNAs of E. dispar and E. histolytica trophozoites isolated using an RNeasy mini kit (Qiagen) were used for cDNA synthesis with a GeneAmp RNA PCR kit (Applied Biosystems). Reaction mixtures for quantitative real-time PCR analysis were prepared using SYBR Premix Ex Taq (Takara), specific primers, Rox dye, and the cDNAs. The primers used were as follows: 5′-TGA CAA AGA CAA TAC TTG TAA AAA GTG-3′ (forward) and 5′-ATT ACT AAC ACA TGC ACA TTT TTT GTC-3′ (reverse) for E. dispar Igl1 genes; 5′-TCG ATG AAA ATA ATG TAT GCC AGA AAT-3′ (forward) and 5′-TCA TCA AGG CAA GCA CAT TGA CTG-3′ (reverse) for E. dispar Igl2 genes; 5′-GTT CAC AGG TTG GTG CTT GTA CG-3′ (forward) and 5′-ACA GTA CAT GGC TTT TCT CCG GTA-3′ (reverse) for E. histolytica Igl1 genes; 5′-GAT TCA CAA ACA AAG GAG TGT GCC-3′ (forward) and 5′-GTG CAT TTG AAC CAC TAG CAG CAA-3′ (reverse) for E. histolytica Igl2 genes; and 5′-CCA GCT ATG TAT GTT GGA ATT CAA G-3′ (forward) and 5′-GAT CAA GTC TAA GAA TAG CAT GTG G-3′ (reverse) for actin genes. Forty cycles of amplification with recording of fluorescence intensity in each cycle were performed using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). After initial denaturation at 95°C for 10 sec, a shuttle PCR protocol consisting of denaturation at 95°C for 5 sec and annealing-extension at 60°C for 30 sec was applied. Relative quantitation with data from the ABI PRISM 7700 Sequence Detection System software version 1.7 was performed by the comparative CT method, using the actin gene as an internal standard. The experiments were repeated 3 times, including the steps of culture of trophozoites and isolation of RNA.
Expression of recombinant Igl1 and Igl2
DNA fragments encoding full-length Igls, except for the N-terminus and C-terminus signal sequences, were obtained by PCR amplification of cloned Igl genes. Primers EdIgl1-S14-Xho (5′-CCC TCG AGG AGT ACA AAG CTG ATA AAC T-3′) and EdIgl-AS-Xho (5′-CCC TCG AGT TAA ATT CCT TTA CTT CCA TT-3′) were used for amplification of the Igl1 gene of SAW1734RclAR. For amplification of the Igl2 gene of SAW1734RclAR, primers EdIgl2-S14-Xho (5′-CCC TCG AGG ATT ACA AAG CTG ATA AAC TCA TC-3′) and EdIgl-AS-Xho were used. PCR was performed as previously described (Tachibana et al. Reference Tachibana, Cheng, Masuda, Horiki and Takeuchi2004). Each amplified DNA fragment was digested with XhoI, purified, and ligated with pET19b vector (Novagen). The plasmid was introduced into competent Escherichia coli JM109 cells and the direction and sequence of inserts were confirmed. E. coli BL21Star(DE3)pLysS cells (Invitrogen) were transformed with the cloned plasmids. Each clone was cultured in 400 ml of Luria-Bertani medium containing ampicillin until the culture reached an optical density of 0·6 at 600 nm. Isopropyl-β-D-thiogalactopyranoside was added to the cultures at a final concentration of 1 mm, and the cultures were incubated at 37°C for 3 h. Preparation of inclusion bodies and refolding of the proteins were performed as previously described (Tachibana et al. Reference Tachibana, Cheng, Masuda, Horiki and Takeuchi2004).
Production of specific mAbs
MAbs to Igl1 and Igl2 of E. dispar SAW1734RclAR were prepared as follows. Six-week-old male BALB/c mice were inoculated intraperitoneally with 10 μg of recombinant proteins in Freund's complete adjuvant and were inoculated again after 2 weeks. After an additional 3 weeks, the mice received only recombinant proteins. Four days later, spleen cells of immunized mice were isolated and fused with X63 Ag8.653 mouse myeloma cells using 50% polyethylene glycol 1500. Hybridomas secreting mAbs against E. dispar Igls were screened by immunofluorescent staining and ELISA, and were cloned by limiting dilution. Immunoglobulin isotypes of mAbs were determined by immunofluorescent staining using subtype-specific antibodies. Ascites was obtained by intraperitoneal inoculation of hybridomas into pristine-primed mice, and immunoglobulin was purified using an Affi-Gel protein A MAPS II kit (Bio-Rad).
Dot blot analysis
Recombinant Igls and sonicated trophozoites of E. dispar SAW1734RclAR were blotted on the nitrocellulose membrane using a Bio-Dot microfiltration apparatus (Bio-Rad). Filter strips were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and reacted with mouse anti-E. dispar Igl mAbs for 30 min. After being washed with PBS containing 0·05% Tween-20 (PBS-Tween), the strips were incubated with horseradish peroxidase (HRP)-labelled goat anti-mouse IgG antibody (MP Biomedicals) for 30 min. The strips were then washed with PBS-Tween and developed with a Konica Immunostaining HRP-1000 kit.
SDS-PAGE and Western blot analysis
Recombinant Igl proteins or E. dispar trophozoites were treated with Laemmli's sample buffer (Laemmli, Reference Laemmli1970) containing 2 mm phenylmethylsulfonyl fluoride, 2 mm N-α-p-tosyl-l-lysine chloromethylketone, 2 mm p-hydroxymercuriphenylsulfonic acid, and 4 μm leupeptin for 5 min at 95°C and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed as previously described (Tachibana et al. Reference Tachibana, Cheng, Masuda, Horiki and Takeuchi2004).
Flow cytometry
Immunophenotypic surface staining of Igl1 and Igl2 using flow cytometry was performed on trophozoites of E. dispar SAW1734RclAR strain. Intact cells were incubated on ice with 3% BSA in PBS for 15 min, and then with a mixture of mAbs ED1-13 and ED2-1 for 15 min. After washing with ice-cold PBS, the cells were incubated with a mixture of Alexa Fluor 488-labelled goat anti-mouse IgG2b-specific antibody (Molecular Probes) and PE-labelled goat anti-mouse IgG1-specific antibody (Santa Cruz Biotechnology) for 15 min on ice. The cells were washed with ice-cold PBS and then fixed in 4% paraformaldehyde. Aliquots of approximately 5000 cells per sample were analysed using FACS Calibur (Becton Dickinson), with data analysis using CellQuest Software (BD Immunocytometry systems).
Confocal microscopy
E. dispar SAW1734RclAR trophozoites were fixed with 4% paraformaldehyde in PBS and attached to silane-coated glass slides using Shandon Cytospin 2. After washing with PBS, the glass slides were incubated with 10% sucrose in PBS for 1 h and then stored at −80°C until use. For double staining of Igl1 and Igl2, fixed trophozoites on the slides were treated with 0·1% Triton X-100 in PBS for 5 min, blocked with 3% BSA in PBS for 30 min and then incubated for 1 h at room temperature with a mixture of 2 mAbs, ED1-13 and ED2-1. After washing, the slides were incubated with a mixture of Alexa Fluor 488-labelled goat anti-mouse IgG2b-specific antibody and Alexa Fluor 594-labelled goat anti-mouse IgG1-specific antibody (Molecular Probes) for 1 h. The stained trophozoites were mounted using glycerol containing 1·25 mg/ml 1,4-diazbicyclo(2,2,2)octane and 10% PBS, and the samples were observed using a Zeiss LSM510 META confocal laser scanning microscope.
RESULTS
Cloning of genes encoding E. dispar Igl1 and Igl2
Two Igl genes cloned from the cDNA library from the E. dispar SAW1734RclAR strain encoded proteins of 1110 and 1106 amino acids, respectively, with calculated molecular masses of 120·9 kDa and 120·3 kDa and theoretical pI values of 5·5 and 4·87, respectively (DDBJ, EMBL, and GenBank Accession numbers AB287423 and AB287424). Two Igl genes were also cloned from the genomic DNA of the E. dispar CYNO9:TPC strain. These genes also encoded proteins of 1110 and 1106 amino acids, respectively, with calculated molecular masses of 121·0 kDa and 120·4 kDa and theoretical pI values of 5·41 and 4·74, respectively (DDBJ, EMBL, and GenBank Accession numbers AB287425 and AB287426). Based on the similarity of the pI values to those of Igls of E. histolytica (5·52 for Igl1 and 5·17 for Igl2) (Cheng et al. Reference Cheng, Hughes, Huston, Loftus, Gilchrist, Lockhart, Ghosh, Miller-Sims, Mann, Petri and Tachibana2001), the former protein was designated as Igl1 and the latter as Igl2 in both E. dispar strains. Multiple alignments of amino acid sequences among these Igl proteins and other E. histolytica Igls are shown in Fig. 1; the amino acid identity between E. dispar Igl1 and Igl2 was 79% in both strains. Differences in amino acids between the proteins in the two E. dispar strains were greater for Igl2 than for Igl1. In comparison with the E. histolytica Igl isoforms, the amino acid sequence identities were 75–76% for Igl1 and 73–74% for Igl2. Insertions of 6 amino acids in Igl1 and Igl2 are present in the two E. dispar strains around position 840. However, all cysteine residues found in E. histolytica Igl proteins were conserved in the E. dispar proteins. Both E. dispar Igl isoforms also contained hydrophobic amino- and carboxy-terminal signal sequences consistent with a GPI-anchored plasma membrane protein, and all Igl sequences contained a signature epidermal growth factor-like domain close to the C-terminus. The nucleotide sequence identities were 88% between the E. dispar Igl1 and Igl2 genes, 83% between the E. dispar and E. histolytica Igl1 genes, and 82% between the E. dispar and E. histolytica Igl2 genes.
Fig. 1. Alignment of the deduced amino-acid sequences of the Igl1 and Igl2 genes from Entamoeba dispar SAW1734RclAR (Ed.SAW1734), E. dispar CYNO9:TPC (Ed.CYNO9) and E. histolytica HM-1:IMSS (Eh.HM1). Identical and conserved amino-acid residues are highlighted in black and grey, respectively.
A BLAST search of E. dispar SAW760 strain genomic sequences <http://www.ncbi.nlm.nih.gov/BLAST/>was performed, although the E. dispar genome project is incomplete. An identical sequence to that of the Igl1 gene of the SAW1734RclAR strain was found (Genbank Accession no. AANV01000026), and 2 sequences (AANV01000644 and AANV01001389) were identified that showed 99% identity with the Igl2 gene of SAW1734RclAR in partially overlapping regions.
Southern blot analyses of Igl genes
Southern blot hybridization using a 420-bp PCR product as a probe was performed on E. dispar SAW1734RclAR genomic DNA digested with DraI, TaqI and HindIII (Fig. 2). The results indicated the presence of 2 Igl genes in the E. dispar genomic DNA.
Fig. 2. Southern blot analysis of Igl genes in Entamoeba dispar SAW1734RclAR. Genomic DNA was digested with DraI (D, lane 1), TaqI (T, lane 2) and HindIII (H, lane 3) and hybridized with the probe. The blot is representative of 2 independent experiments. Numbers to the right indicate the sizes of DNA markers (in kilobases).
Real-time RT-PCR analysis of Igl genes
Expression levels of Igl1 and Igl2 are compared in Fig. 3. In the SAW1734RclAR strain, expression of Igl1 was 3 times higher than that of Igl2 (P<0·001), and in the CYNO9:TPC strain, expression of Igl1 was 5 times higher than that of Igl2 (P<0·01). Higher expression of Igl1 compared to Igl2 was also observed in E. histolytica (P<0·001). Expression of Igl1 was lower in E. dispar than in E. histolytica (SAW1734RclAR vs HM-1:IMSS, P<0·01; CYNO9:TPC vs HM-1:IMSS, not significant), whereas Igl2 expression was similar in the two species.
Fig. 3. Real-time reverse transcription PCR analysis of Igl genes from Entamoeba dispar and E. histolytica. Expression levels of Igl1 (open bars) and Igl2 (filled bars) in trophozoites from E. dispar SAW1734RclAR, E. dispar CYNO9:TPC and E. histolytica HM-1:IMSS are expressed as values relative to the expression level of actin. Vertical bars indicate the s.e. of the mean from 3 experiments.
Dot blot and Western blot analyses of Igls
Full length (except for the signal sequence) recombinant E. dispar Igls from the SAW1724RclAR strain were prepared in Escherichia coli. The apparent molecular weight of the recombinant protein with the leader peptide was slightly larger for Igl2 (190 kDa) than for Igl1 (170 kDa) in SDS-PAGE under reducing conditions (data not shown). Recombinant Igl1 and Igl2 proteins were used for immunization of mice to prepare mAbs specific for each Igl. In dot blot analysis, mAb ED1-13 reacted specifically with recombinant Igl1, but not with recombinant Igl2; in contrast, mAb ED2-1 was reactive specifically with recombinant Igl2 (Fig. 4A). In conditions under which the reactivity of these mAbs to E. dispar trophozoites was comparable, differences in reactivity to equal amounts of recombinant Igls were observed. The reactivity of mAb ED1-13 to 104 trophozoites was similar to the reactivity to 1 μg of EdIgl1, whereas the reactivity of mAb ED2-1 to 104 trophozoites were comparable to the reactivity to 0·1 μg of EdIgl2, suggesting that the amount of Igl2 was approximately one-tenth that of Igl1 in the trophozoites. In Western blot analysis using trophozoites from the SAW1734RclAR strain, mAbs ED1-13 and ED2-1 recognized a 100 kDa and a 120 kDa band, respectively, under non-reducing conditions (Fig. 4B). No bands were detected in the Western blot under reducing conditions, indicating that the mAbs recognized discontinuous epitopes on the Igl proteins.
Fig. 4. (A) Reactivity of anti-Igl monoclonal antibodies to recombinant Igls and crude antigens of Entamoeba dispar in dot blot analysis. Various concentrations (0·01, 0·1 and 1 μg) of recombinant Igl1 (rIgl1) and recombinant Igl2 (rIgl2) and various numbers (102, 103 and 104) of sonicated trophozoites from strain SAW1734RclAR were spotted on nitrocellulose membranes. One strip was stained with Coomassie brilliant blue (CBB). Other strips were treated with anti-E. dispar Igl monoclonal antibodies (ED1-13, ED2-1) and anti-E. histolytica Igl monoclonal antibody (EH3015). HRP-conjugated goat antibody to mouse IgG was used as a secondary antibody. The blot is representative of 2 independent experiments. (B) Western immunoblot analysis of native Igls of E. dispar. Lysates of SAW1734RclAR trophozoites were subjected to SDS-PAGE in a 7·5% gel under non-reducing conditions and transferred to polyvinylidene difluoride membranes. Protein bands of the size marker in lane 1 were stained with Coomassie brilliant blue. The strips were treated with monoclonal antibodies as follows: lane 2, ED1-13; lane 3, ED2-1; and lane 4, EH3015. HRP-conjugated goat antibody to mouse IgG was used as a secondary antibody. The blot is representative of 3 independent experiments. The numbers to the left indicate molecular masses (in kilodaltons).
Phenotypic expression of Igls on the surface of trophozoites
To compare the amounts of Igl1 and Igl2 expressed on the surface of trophozoites from the E. dispar SAW1724RclAR strain, flow cytometric analysis was performed using specific mAbs for each Igls (Fig. 5). The results demonstrated that almost all trophozoites expressed both Igl1 and Igl2 on the cell surface.
Fig. 5. Flow cytometric analysis of phenotypic expression of Igl1 and Igl2 on the surface of Entamoeba dispar trophozoites. Intact trophozoites from the SAW1734RclAR strain were double stained with monoclonal antibodies ED1-13 (specific for Igl1, IgG2b) and ED2-1 (specific for Igl2, IgG1), followed by Alexa Fluor 488-labelled goat anti-mouse IgG2b-specific antibody and PE-labelled goat anti-mouse IgG1-specific antibody (A). The control was stained only with secondary antibodies (B). The results are representative of 3 independent experiments.
Localization of Igls on trophozoites
Localization of Igl1 and Igl2 on E. dispar trophozoites was examined by confocal laser scanning microscopy using specific mAbs. Both Igls were localized on the plasma membrane and in cytoplasm in all trophozoites (Figs 6 and 7). However, the amount of each Igl in the trophozoites was variable, especially in the cytoplasm; that is, Igl1- and Igl2-dominant cells were present (arrows and arrowheads in Fig. 6). In addition, Igl1- and Igl2-dominant vacuoles were also observed within a single trophozoite (arrow and arrowhead in Fig. 7).
Fig. 6. Localization of Igl1 and Igl2 on trophozoites of Entamoeba dispar SAW1734RclAR observed by confocal laser scanning microscopy. Fixed trophozoites were stained with ED1-13 specific for Igl1 and a secondary Alexa Fluor 488-labelled anti-mouse IgG2b antibody (green) (A) or ED2-1 specific for Igl2 and a secondary Alexa Fluor 594-labelled anti-mouse IgG1 antibody (red) (B). Differential interference contrast microscopy is shown in (C). A merged image is shown in (D). Arrows and arrowheads indicate Igl1- and Igl2-dominant cells, respectively.
Fig. 7. Stereo images of localization of Igl1 (A) and Igl2 (B) on Entamoeba dispar trophozoites observed by confocal laser scanning microscopy. Fixed trophozoites were stained as described in Fig. 6. Differential interference contrast microscopy is shown in (C). A merged image is shown in (D). Arrow and arrowhead indicate single localization of Igl1 and Igl2, respectively.
DISCUSSION
Comparison of the 2 Igl genes cloned from E. dispar with those from E. histolytica indicated differences in the sequences of the corresponding proteins in E. dispar and E. histolytica and in the 2 strains of E. dispar. However, all the cysteine residues, including the CXXC motifs, were conserved between species and between strains, which suggests that the fold of the protein is maintained and is important for its function. Amino acid identities of Igl1 and Igl2 within species (79% in E. dispar and 81% in E. histolytica; Cheng et al. Reference Cheng, Hughes, Huston, Loftus, Gilchrist, Lockhart, Ghosh, Miller-Sims, Mann, Petri and Tachibana2001) were higher than those for each Igl between the 2 species (75–76% for Igl1 and 73–74% for Igl2). Insertions of 6 amino acids around position 840 in E. dispar were present in both Igl1 and Igl2, as shown in Fig. 1. Nucleotide identities of the 2 Igl genes were also higher within species, compared to the respective identities of the Igl genes between E. dispar and E. histolytica. These results suggest that duplication of the genes may have occurred after divergence of the species.
The properties of the Gal/GalNAc lectin of E. histolytica were demonstrated in a 150 kDa fraction purified by affinity chromatography using the E. histolytica-specific mAb EH3015 (Cheng et al. Reference Cheng, Tsukamoto, Kaneda and Tachibana1998). In addition, Igl1 and Igl2 of E. histolytica have been detected, in addition to Hgl and Lgl, in the protein fraction that binds specifically to GalNAc-BSA-coated magnetic beads (McCoy and Mann, Reference McCoy and Mann2005). However, when we performed a preliminary examination of the reactivity of recombinant Igls from E. histolytica and E. dispar with GalNAc27-BSA by dot blot analysis and surface plasmon resonance, we could not prove that the recombinant proteins had sugar-binding properties (data not shown). Therefore, the Igls may exist as part of the lectin complex, perhaps with non-covalent association to another protein containing a sugar-binding site, with this association occurring either directly or being mediated by a third protein. Recently, it has been reported that Igl of E. histolytica is found in the protein fraction that interacts with purified brush border from human enterocytes (Seigneur et al. Reference Seigneur, Mounier, Prevost and Guillen2005). It has also been demonstrated that the 140 kDa fibronectin-binding molecule of E. histolytica (Talamas-Rohana et al. Reference Talamás-Rohana, Rosales-Encina, Gutierrez and Hernández1992) is identical with Igl2 (Hernandez-Ramirez et al. Reference Hernández-Ramírez, Rios, Angel, Magos, Pérez-Castillo, Rosales-Encina, Castillo-Henkel and Talamás-Rohana2007). These observations indicate that Igls are important proteins for amoebic adherence to host cells, and since Igls are also expressed in the non-pathogenic amoeba, it seems likely that these proteins are important for colonization of amoebae in the large intestine.
One of the interesting observations in this study is the difference in expression between Igl1 and Igl2. Since higher expression of Igl1 was observed at both protein and mRNA levels, the difference between the isoforms seems to be regulated mostly at the transcriptional level. The expression level of Igl1 was also lower in E. dispar than in E. histolytica, whereas that of Igl2 was comparable in the two species. It has been demonstrated that expression of Hgl (Hgl2) and Lgl (Lgl1) in E. dispar is lower than in E. histolytica (Pillai et al. Reference Pillai, Britten, Ackers, Ravdin and Kain1997, Reference Pillai, Kobayashi and Kain2001); therefore, Igl1 may be more closely associated with Hgl and Lgl. However, it is unknown whether the 2 Igl isoforms are associated with different isoforms of Hgl or Lgl. DNA microarray analyses have shown that a large number of genes are expressed differently in E. histolytica and E. dispar, and that there is a difference in gene expression between strains of E. histolytica of high and low virulence (Shah et al. Reference Shah, MacFarlane, Bhattacharya, Matese, Demeter, Stroup and Singh2005; MacFarlane and Singh, Reference MacFarlane and Singh2006; Davis et al. Reference Davis, Schulze and Stanley2007). Lower expression of Hgl genes in E. dispar compared to E. histolytica has also been confirmed (MacFarlane and Singh, Reference MacFarlane and Singh2006), but Lgl3 expression was found to be higher in an E. histolytica strain of low virulence compared to a strain of high virulence (Davis et al. Reference Davis, Schulze and Stanley2007). Igl genes are down-regulated by heat shock stress, as are most Hgl and Lgl genes (Weber et al. Reference Weber, Guigon, Bouchier, Frangeul, Moreira, Sismeiro, Gouyette, Mirelman, Coppee and Guillen2006). Collectively, these observations suggest that the lectin as a whole is important for adherence and subsequent pathogenesis.
We also compared phenotypic expression of the two Igls in each trophozoite using flow cytometry and confocal microscopy. Interestingly, some trophozoites expressed Igl1 or Igl2 dominantly, although most cells expressed the two Igl proteins to a comparable extent, suggesting that both Igls are important for the amoeba. Igls were also localized in intracellular vacuoles of E. dispar. Recently, it has been demonstrated that Igl is contained in phagosomes of E. histolytica and that the quantity of Igl varies during phagosome maturation (Okada et al. Reference Okada, Huston, Mann, Petri, Kita and Nozaki2005, Reference Okada, Huston, Oue, Mann, Petri, Kita and Nozaki2006). The different localization of Igl1 and Igl2 in vacuoles suggests that the functions of the two Igls may differ or that their expression may vary during maturation of phagosomes or depending on certain cellular conditions. However, there may also be differences in the involvement of microtubules and proteases in phagosome maturation and degradation in E. histolytica and E. dispar (Mitra et al. Reference Mitra, Yasuda, Kobayashi, Saito-Nakano and Nozaki2005). In E. histolytica, different subcellular localization of the two Igl isoforms has yet to be shown.
In conclusion, this is the first study of the differences between Igl1 and Igl2 of E. dispar. Igl seems to be a vaccine candidate for amebiasis and may also be a useful antigenic molecule for specific serodiagnosis of amoebiasis (Cheng and Tachibana, Reference Cheng and Tachibana2001; Tachibana et al. Reference Tachibana, Cheng, Masuda, Horiki and Takeuchi2004). Therefore, further studies of Igl are required to clarify its role in the host-parasite relationship.
This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science, and a grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare of Japan. X.-J. C. is a recipient of a Postdoctoral Fellowship for Foreign Researchers from the Japanese Society for the Promotion of Science.
