Expression in fibroblasts and in live animals of Entamoeba histolytica polypeptides EhCP112 and EhADH112

Xochil Madriz1,2, Máximo B. Martínez2, Mario A. Rodríguez3, Gustavo Sierra2, Carolina Martínez-López1, Ana M. Riverón4, Leopoldo Flores3 and Esther Orozco3

1 Escuela Nacional de Medicina y Homeopatía, IPN, Guillermo Massieu Helguera #239, DF 07320, Mexico
2 División de Biología Molecular, Instituto Finlay, AP 16017 Ciudad de la Habana, C.P. 11600, Cuba
3 Departamento de Patología Experimental, CINVESTAV, IPN, AP 14-740, DF 07000, Mexico
4 Departamento de Biología Molecular, CENIC, AP 6690 Ciudad de La Habana, Cuba

Correspondence
Esther Orozco
esther{at}mail.cinvestav.mx


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved in Entamoeba histolytica's cytopathic effect, target-cell adherence and phagocytosis. The EhCPADH complex is located in the plasma membrane and cytoplasmic vacuoles. Here, the independent expression of EhCP112 and EhADH112 in fibroblasts and hamsters was analysed. Also investigated was the immunological response in animals independently inoculated with plasmid pcDNA-Ehcp112, which carries the complete cysteine protease-encoding gene, or with plasmid pcDNA-Ehadh112, which carries the C terminus of the adhesin-encoding gene, or with a mixture of both. Both proteins were expressed in the plasma membranes of the transfected fibroblasts. EhCP112 was toxic for the mammalian cells. Proteins were also independently expressed in hamsters after inoculation with the plasmids. Their expression was indirectly evaluated by the presence of antibodies in the inoculated animals. Remarkably, co-immunization of the animals with the two DNA plasmids resulted in an earlier and higher anti-E. histolytica IgG induction than immunization with separate plasmids. In contrast, the cellular immune response was not noticeably improved by the plasmid mixture. Interestingly, protection against liver abscesses was detected only in animals that received the plasmid mixture and no protection was observed in hamsters independently inoculated with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112.


Abbreviations: EhADH112, adhesin; EhCP112, cysteine protease; RBC, red blood cell


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Entamoeba histolytica, the protozoan responsible for human amoebiasis, provokes up to 100 000 deaths each year and is the third major cause of death attributed to parasites worldwide (Walsh, 1986). Morbidity and mortality associated with this infection persist in many poor countries. Intact trophozoites, trophozoite lysates, specific antigens and recombinant proteins protect animals against liver abscesses (Ghadirian et al., 1980; Soong et al., 1995a, b; Vinayak et al., 1987, 1980), but we do not yet have an efficient vaccine for human amoebiasis. This is due mainly to the fact that we ignore very many of the E. histolytica protein–protein interactions and the effects they have, along with the trophozoites, on producing damage in the host.

Two proteins have been mainly used in protection experiments: the galactose/N-acetylgalactosamine inhibitable lectin (Petri et al., 1987) and the serine-rich protein (Stanley et al., 1990), both of which are involved in adherence. Immunization experiments using DNA plasmids carrying genes encoding these proteins have also been done and have shown that these DNAs induce an immune response to E. histolytica (Gaucher & Chadee, 2002; Zhang & Stanley, 1999). Immunization with DNA produces a broad-spectrum and long-lasting immune response, better cross-strain protection and a reduced cost in terms of production of the vaccine (Kowalczyk & Ertl, 1999). In addition, the unmethylated CpG motifs in the backbone of DNA vectors elicit secretion of cytokines by lymphocytes, including IL-12 and TNF-{alpha} (Kowalczyk & Ertl, 1999), which promote the development of the Th1 immune response (Campbell & Chadee, 1997).

EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved in E. histolytica's cytopathic effect, adhesion and phagocytosis (Arroyo & Orozco, 1987; Garcia-Rivera et al., 1999; Rodriguez et al., 1989). The EhCPADH complex has a molecular mass of 124·6 kDa and is located in the plasma membrane and cytoplasmic vacuoles (Garcia-Rivera et al., 1999). Our previous results have shown that hamsters immunized with a recombinant fragment of EhCPADH (Garcia-Rivera et al., 1999) were partially protected against liver abscesses (Martinez-Lopez et al., 2004). Two adjacent genes encode EhCPADH (Garcia-Rivera et al., 1999). The Ehcp112 gene encodes a 446 aa polypeptide with 27–31 % identity to other cysteine proteases. EhCP112 contains a putative transmembrane sequence (aa 395–416) and an integrin attachment domain (RGD) (Garcia-Rivera et al., 1999). The Ehadh112 gene encodes a 687 aa polypeptide (EhADH112), which contains three predicted transmembrane segments (aa 114–130, 176–195 and 224–247) and the cell-adherence domain located at the carboxy terminal region (Garcia-Rivera et al., 1999). The monoclonal antibody (mAb) anti-EhCPADH inhibits trophozoite adherence and recognizes an epitope in the carboxy domain of EhADH112 with homology to another epitope located in the EhCP112 polypeptide (Garcia-Rivera et al., 1999). Further characterization of the EhCPADH complex is important to better understand the functions of both of its polypeptides.

In this work, we studied the expression of EhCP112 and EhADH112 after plasmids encoding them had been used independently to transfect fibroblasts and to inoculate hamsters. Both proteins were expressed on the fibroblasts' surface and both were expressed in the animals inoculated with the plasmids, measured by their immune response against E. histolytica trophozoites.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
E. histolytica and cell cultures.
E. histolytica trophozoites (strain HM1 : IMSS; kindly provided by Dr Ruy Pérez Tamayo, Facultad de Medicina, UNAM, Mexico) were passed multiple times through the liver of hamsters to maintain their virulence. The strain was axenically cultured in TYI-S-33 medium and harvested during the exponential growth phase (Diamond et al., 1978). L929 murine fibroblasts were cultured in RPMI medium (GIBCO) supplemented with 10 % fetal calf serum (FCS; GIBCO) at 37 °C in 5 % CO2. All assays presented here were done at least three times.

Construction of plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 and transfection of L929 fibroblasts.
The full-length Ehcp112 gene (1338 bp) and a 1755 bp DNA fragment of the Ehadh112 gene, lacking 306 bp at the 5' end, were independently cloned in the BamHI/EcoRI and XbaI/ApaI sites of the vector pcDNA3 (Invitrogen), respectively (Fig. 1). Constructs were checked by sequencing using the dideoxynucleotide chain-termination method (Sanger et al., 1977). The plasmids were purified by using Qiagen Giga-Plasmid columns. Five micrograms of each plasmid were incubated with 25 µg lipofectAmine (GIBCO) in a final volume of 2·4 ml during 25 min at room temperature, following the manufacturer's instructions. The mixture was added to the L929 culture cells at 70 % confluence and the plates with the cells were incubated at 37 °C in 5 % CO2 for 5 h. After that, medium was supplemented with 20 % FCS. Geneticin (GIBCO) [1 mg (ml medium)–1] was added to the cells 48 h after transfection. For other experiments, the complete Ehcp112 gene and 729 bp at the carboxy terminus of the Ehadh112 gene were cloned in plasmid pRST A and recombinant proteins were produced in IPTG-induced Escherichia coli BL21(DE3) (Garcia-Rivera et al., 1999).



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Fig. 1. Schematic representation of construction of plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112. (a) Genome organization of the Ehcp112 and Ehadh112 genes of E. histolytica. NCR, non-coding region. (b) Vector pcDNA3. MCS, multi-cloning site; SV40ori, replication origin of the SV40 virus; neor, neomycin-resistance gene; ColE1, replication origin of ColE1 plasmids; ampr, ampicillin-resistance gene; pCMV, cytomegalovirus promoter. (c, d) Plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112. (e, f) EhCP112 and EhADH112 polypeptides. SP, signal peptide; Pp, pro-peptide; RGD, integrin attachment domain; TM, transmembrane sequences; mAb recognition, amino acid sequence in which is located the epitope recognized by the anti-EhCPADH mAb.

 
Confocal immunofluorescence.
The transfected L929 cells were grown on cover-slides introduced into 60 mm Petri dishes. Cells were fixed by using 3·8 % paraformaldehyde (Sigma) for 2 h at 37 °C, washed with PBS, permeabilized with 0·2 % Triton X-100 (Sigma) for 2 min, washed again with PBS and then incubated for 1 h at 37 °C with the mAb anti-EhCPADH (Arroyo & Orozco, 1987) (titre 1 : 10 in 1 % BSA). Washed cells were incubated for 30 min at 37 °C with FITC-labelled goat anti-mouse antibodies (1 : 200), washed and observed through a Nikon Diaphot 200 microscope attached to a laser confocal scanning system, MRC 1024 (Bio-Rad).

Inoculation of hamsters with plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112.
Syrian golden hamsters (Mesocricetus auratus) (male, 4 weeks old) were divided into groups of eight animals and intradermally injected at the base of the tail with 50, 100 or 200 µg of pcDNA-Ehcp112 or pcDNA-Ehadh112 or with a mixture of 50 µg of both plasmids. As a control, we injected animals with 100 µg of pcDNA3. Animals were immunized on days 0, 20 and 40, and were bled from the retro-orbital plexus on days 0, 10, 20, 30, 40 and 50. At day 57, after three inoculations, hamsters were challenged with 1·5x105 trophozoites, inoculated via the portal vein, as described below. Animals were bled and killed under anaesthesia (6·3 mg Anaesthesal ml–1) (Pfizer) 1 week after challenge (day 64).

ELISA.
ELISA plates (96-well; Costar) (Engvall & Perlman, 1971) were coated with heat-fixed trophozoites (52 °C, 15 min) resuspended in PBS (104 per well) as described by Zhang & Stanley (1996). Then, 50 µl of the hamster sera (1 : 500) were added to the wells. Plates were incubated overnight at 4 °C, washed three times with PBS and incubated for 2 h at 37 °C with 50 µl of horseradish peroxidase (HRP)-labelled goat anti-hamster IgG (1 : 2000) (Cappel). The enzymic reaction was developed as described by Engvall & Perlman (1971); the colour developed was measured in an ELISA reader (ICN, MS2) at OD492.

SDS-PAGE and Western blot assays.
Trophozoites were washed twice with PBS at 4 °C and disrupted by freezing–thawing in the presence of 20 mM p-hydroxymercuribenzoate (Sigma). Samples were electrophoresed on SDS-PAGE (12 % polyacrylamide) gels and transferred to nitrocellulose membranes (Towbin et al., 1979), which were blocked with 2 % non-fat milk in 0·5 % Tween 20 in PBS for 1 h. Membranes were incubated with the hamster anti-sera (1 : 100) or with the anti-EhCPADH mAb overnight at 4 °C, and then for 1 h at room temperature with HRP-labelled goat anti-hamster IgG (Cappel) or goat anti-mouse IgM (Zymed), respectively, and developed with 4-chloro-1-naphthol and 0·04 % H2O2.

In vitro cell proliferation assay.
Hamsters were immunized and challenged with live trophozoites as described above. Seven days after challenge (day 64), animals were killed and their spleens were placed in 5 ml of serum-free RPMI medium. The lymphocytes were obtained and resuspended at 2x106 (ml RPMI medium)–1 supplemented with 10 % heat-inactivated FCS, 100 U penicillin ml–1 and 100 µg streptomycin sulphate ml–1. For T-cell proliferation, 2x105 lymphocytes per well were cultured with 1x104 or 4x104 heat-killed trophozoites (52 °C, 15 min). As a negative control, lymphocytes were incubated with serum-free RPMI medium; as a positive control, lymphocytes were incubated with 2·5 µg ml–1 concanavalin A (Pharmacia) per well. Cells were cultured for 48 h at 37 °C in a humid atmosphere containing 5 % CO2. Then, 1 µCi (37 kBq) per well of [3H]thymidine (Amersham Pharmacia) was added to the cultures for an additional 18 h. Incorporated radioactivity was measured by liquid scintillation counting (Beckman Instruments LS5801) in cells washed and filtered through a glass filter paper for cell harvest (Nunc).

Adherence inhibition assays using hamster antisera.
Trophozoites (6x105) were pre-incubated at 4 °C for 20 min with 1·2 µl immune hamster sera adjusted to the same titre and diluted 1 : 500 in serum-free TYI-S-33 medium. Then, 600 µl of human red blood cells (RBCs) (1x108 cells ml–1) were added and cell mixtures were incubated for 10 min at 4 °C. RBCs adhered to 100 trophozoites were counted as described previously (Garcia-Rivera et al., 1982). The adherence efficiency was calculated taking as 100 % the RBCs adhered to trophozoites that were incubated in the absence of serum. As controls, we used sera from hamsters inoculated with pcDNA3 or with the pre-immune sera.

Virulence in vivo.
Under sterile conditions and intra-peritoneal anaesthesia (6·3 mg Anaesthesal ml–1), groups of eight hamsters for each condition were laparotomized and intraportally infected with 1·5x105 trophozoites in a volume of 0·1 ml PBS using a 29 gauge needle (Tsutsumi et al., 1984). Seven days after the challenge, the animals were anaesthetized, their livers were removed and the number and size of the abscesses were recorded.

Statistical analysis.
Collected data were submitted to ANOVA, and the less statistical difference (LSD) test was used to determine significant differences between the experimental groups. The probability level for significance was 95 %. All values are expressed as mean±SD.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112
The genes Ehcp112 and Ehadh112 encode the polypeptides that form the E. histolytica EhCPADH protein complex (Fig. 1a, e, f). The putative polypeptide encoded by the Ehcp112 gene cloned in pcDNA3 (Fig. 1b, c) contains the 19 aa of the signal peptide, the 117 aa sequence of the pro-peptide and 310 aa of the mature cysteine protease (Fig. 1e). The whole protein has a predicted molecular mass of 49 kDa. According to the TMPRED program (Hofmann & Stoffel, 1993), mature EhCP112 has a putative transmembrane sequence which may sort the protein to the plasma membrane, the RGD domain which might allow the protein to interact with the cell integrins (Fig. 1e) and an epitope recognized by the anti-EhCPADH mAb (Garcia-Rivera et al., 1999). We also cloned a 1755 bp fragment of the Ehadh112 gene, which lacked 306 bp at the 5' end, to generate plasmid pcDNA-Ehadh112 (Fig. 1a, d), which encodes a 585 aa polypeptide with a predicted molecular mass of 64·3 kDa. The TMPRED program (Hofmann & Stoffel, 1993) predicts that this polypeptide has three putative transmembrane sequences. It also contains the adherence domain identified in the EhADH112 protein by the anti-EhCPADH mAb (Fig. 1f) (Garcia-Rivera et al., 1999, Martinez-Lopez et al., 2004).

Plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 are expressed in the plasma membrane of mammalian cells
We investigated whether plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 were able to express in mammalian cells and the protein location in transfected cells. L929 fibroblast cells transfected with each one of the plasmids and grown in medium supplemented with neomycin were examined every day. After 4 days, all the non-transfected cells died due to the antibiotic, whereas cells transfected with either of the plasmids survived. Cells transfected with Ehcp112 (Ehcp112-L929 cells) and Ehadh112 (Ehadh112-L929 cells) presented differences. Whereas the Ehcp112-L929 cells appeared rounded and detached from the plate and many of them died, Ehadh112-L929 cells seemed to be always healthy. The appearance of the Ehcp112-L929 cells was similar to that presented by cultured cell monolayers incubated with trophozoite extracts. The effect may be due to the presence of mature EhCP112 in the cells, which is toxic for mammalian cells. One week after the experiments, the Ehcp112-L929 cells showed only 10 % survival. However, a few days later, the surviving cells divided and grew well. In contrast, 75–85 % of the cells transfected with pcDNA-Ehadh112 survived. We confirmed the expression of the E. histolytica proteins in the Ehcp112-L929 and Ehadh112-L929 cells by immunofluorescence using the anti-EhCPADH mAb (Fig. 2a, b), which recognizes common epitopes in the EhCP112 and EhADH112 polypeptides. We have not precisely identified the common epitopes recognized by the mAb, but EhCP112 and EhADH112 share sequences in their C terminus region that our preliminary phage display experiments suggest are recognized by the mAb (Fig. 2, bottom). The anti-EhCPADH mAb reacted with the surface of the Ehcp112-L929 and Ehadh112-L929 cells (Fig. 2b). In all experiments, 99 % of the cells gave fluorescence, although intensity was higher in Ehadh112-L929 than in Ehcp112-L929 cells (Fig. 2b, a). Fluorescence was not detected in the cells transfected with pcDNA3 (Fig. 2d), nor in transfected cells incubated only with the secondary antibody (Fig. 2c). These results indicated that pcDNA3 drove the expression of the cloned genes to produce the EhCP112 and EhADH112 proteins in the mammalian transfected cells. In addition, the recombinant proteins were sorted to the plasma membrane of Ehcp112-L929 and Ehadh112-L929 cells as the native proteins are in live trophozoites (Arroyo & Orozco, 1987).



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Fig. 2. Immunofluorescence of murine L929 cells transfected with pcDNA-Ehcp112 or pcDNA-Ehadh112. L929 cells were transfected with (a) pcDNA-Ehcp112, (b, c) pcDNA-Ehadh112 or (d) pcDNA3. The EhCP112 and EhADH112 polypeptides were identified by the anti-EhCPADH mAb in immunofluorescence assays. (a, b, d) Confocal microscopy of cells incubated with the anti-EhCPADH mAb, then with secondary FITC-conjugated antibodies. (c) Confocal microscopy of cells incubated only with the secondary FITC-conjugated antibodies. Below the images are shown two amino acid sequences of EhCP112 and EhADH112 with high homology.

 
pcDNA-Ehcp112 and pcDNA-Ehadh112 are expressed in hamsters
To investigate whether plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 were also able to express in hamsters, we inoculated the animals with 50, 100 or 200 µg of the plasmids and examined their sera by ELISA to look for antibodies against the proteins. As controls, we used the pre-immune sera of each animal, which were submitted to Western blot assays and selected by their total absence of E. histolytica recognition before plasmid inoculation. The OD492 value given by the pre-immune sera (0·0–0·2) was subtracted from the serum OD492 value given by the corresponding immunized animal. No significant humoral response was detected at days 0, 10, 20, 30 and 40 with any dose or plasmids used. This means that, with the plasmid doses used, the amount of antigen produced requires more than 40 days to generate an immune response in the animals. However, at day 50, after the third immunization, IgG against the trophozoites was found in the sera of animals inoculated with 100 or 200 µg of pcDNA-Ehcp112 (Fig. 3a). Whereas animals inoculated with 50 µg of this plasmid exhibited similar OD492 values to those inoculated only with the vector, the OD492 values obtained from the sera of the different animals inoculated with 100 or 200 µg of pcDNA-Ehcp112 varied due to discrepancies in the IgG titre.



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Fig. 3. Humoral response of animals immunized with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112. Hamsters were immunized with different amounts of pcDNA-Ehcp112 or pcDNA-Ehadh112 and sera were obtained at day 50, after the third immunization. (a, b) IgG against E. histolytica evaluated by ELISA. The data correspond to the mean of the OD492 values from sera (1 : 500) of animals immunized with the same dose (n=8) and normalized with respect to the values of the pre-immune sera at the same dilution. Lines indicate the SD. (c, d) Western blot using total E. histolytica proteins or (e) the recombinant EhCP112 polypeptide or (f) the recombinant EhADH112 polypeptide. Lane 1, sera from animals immunized with pcDNA-Ehcp112 (c, e) or pcDNA-Ehadh112 (d, f). Lane 2, anti-EhCPADH mAb. Lane 3, pre-immune sera. Lane 4, sera from animals inoculated with pcDNA3. M, molecular mass markers. Arrowheads signal the migration site of the EhCPADH protein complex.

 
Immunization with pcDNA-Ehadh112 also generated a humoral response at day 50 (Fig. 3b). Animals gave a better response than those inoculated with pcDNA-Ehcp112. Even hamsters that received 50 µg of pcDNA-Ehahd112 showed a good IgG titre (Fig. 3b). Animals that received plasmid pcDNA-Ehadh112 also presented titre variation (Fig. 3b). Variation in the immune response among the animals inoculated with the same antigen in identical conditions is widely known and it has been reported for different E. histolytica animal models (Soong et al., 1995a, b). All hamsters inoculated only with the vector gave OD492 values ranging from 0 to 0·2 (Fig. 3a, b), indicating that the presence of antibodies against the amoebic antigens was due to the inoculation with the plasmids that were expressed inside the animals. Analysis of the data obtained from independent experiments indicated that the humoral response was significant (P<0·05).

The specificity of the humoral response was investigated by Western blot assays using total E. histolytica proteins and a sera pool from hamsters immunized three times with 100 µg of pcDNA-Ehcp112 or pcDNA-Ehadh112. In both cases, the sera reacted with the EhCPADH complex (Fig. 3c, d, lane 1), which was also recognized by the anti-EhCPADH mAb (Fig. 3c, d, lane 2). The pre-immune sera and the sera from the animals immunized with vector pcDNA3 did not recognize any band (Fig. 3c, d, lane 3, 4), indicating a specific humoral response against the EhCPADH protein complex. Interestingly, the sera from animals inoculated with pcDNA-Ehcp112 or pcDNA-Ehadh112 did not recognize the native EhCP112 (49 kDa) and EhADH112 (75·5 kDa) in total trophozoites proteins, suggesting that the polypeptides form the EhCPADH complex immediately after their synthesis and that the amount of free EhCP112 and EhADH112 is very low to be detected by these antibodies. As expected, the recombinant EhCP112 and EhADH112 proteins produced in bacteria were recognized by the anti-EhCPADH mAb and by the respective immune sera against EhCP112 and EhADH112 polypeptides (Fig. 3e, f). Anti-EhCPADH mAb and the immune sera of the immunized animals with pcDNA-Ehcp112 reacted with 52 and 40 kDa proteins in the lane where the rEhCP112 protein was electrophoresed (Fig. 3e). Fifty-two kilodaltons is the expected molecular mass of the polypeptide according to the cDNA fragment cloned in the vector, and the 40 kDa band may correspond to a degradation product of the whole recombinant EhCP112. In contrast, the anti-EhCPADH mAb and the antibodies of the immunized animals with plasmid pcDNA-Ehadh112 reacted with a 30 kDa band in the lane where is a 30 kDa recombinant fragment of the EhADH112 protein (Fig. 3f) cloned in the expression vector pRSET B (Martinez-Lopez et al., 2004).

pcDNA-Ehcp112 and pcDNA-Ehadh112 induce a cell-mediated immune response in hamsters
We evaluated the cellular immune response in the immunized animals, 1 week after challenging them with virulent trophozoites. Sixty-four days after the first immunization, cell proliferation was measured in spleen lymphocytes cultured in the presence of [3H]thymidine and on 1x104 or 4x104 heat-killed trophozoites per well for in vitro stimulation. No significant variations were found with the two different numbers of trophozoites used. Animals inoculated with 100 µg of pcDNA-Ehadh112 gave a better response (8769–11 352 c.p.m.) than those inoculated with 100 µg of pcDNA-Ehcp112 (2362–2581 c.p.m.). Differences between these two groups were significant in all experiments (P<0·05). The proliferative response of the control animals that were injected only with the vector and challenged with live trophozoites was 578–1009 c.p.m. (Fig. 4a, b).



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Fig. 4. Cellular response of animals immunized with pcDNA-Ehcp112 or pcDNA-Ehadh112. Hamsters were immunized with different amounts of pcDNA-Ehcp112 or pcDNA-Ehadh112. After the challenge with live trophozoites (day 64), lymphocytes from the spleens of immunized animals were obtained and their radioactive incorporation under the stimulation of different amounts of heat-killed trophozoites (open bars, 1x104; solid bars, 4x104) was determined. The data correspond to the mean c.p.m. values obtained from animals immunized with the same dose (n=8). Lines indicate SD.

 
Mixture of plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 improves the immune response in hamsters
To investigate whether the expression of both polypeptides (EhCP112 and EhADH112) could increase the immune response in animals, we used a mixture containing 50 µg of each plasmid. At day 10 after the first inoculation, the level of the IgG antibodies against E. histolytica was higher (OD492 0·2–0·5) than the level of IgG in sera from animals inoculated with vector pcDNA3, analysed at day 50 (OD492 0·01–0·2) (Fig. 5a). The humoral response of the immunized animals augmented at day 20 (0·3–0·8) (Fig. 5a). As expected, differences in the antibody titre among the animals of each group were also observed in these experiments. In Western blot experiments, immune sera recognized in total extracts of trophozoites the EhCPADH complex that was also recognized by the anti-EhCPADH mAb (Fig. 5b, lane 1, 2). In addition to the EhCPADH protein complex, a band of 90 kDa and a strong one of 65 kDa appeared in the membranes. These bands do not have the molecular mass of the native EhADH112 and EhCP112 proteins, but appeared after immunization, and we do not yet know their meaning. The pre-immune sera and the control sera of hamsters immunized with pcDNA3 gave low unspecific signal (Fig. 5b, lane 3, 4), indicating a specific humoral response of the vaccinated animals. In these experiments, certain sera recognized other bands in addition to the EhCPADH complex, probably due to unspecific reaction; this was especially evident when we used a serum pool to reveal the Western blots. Remarkably, although the humoral response augmented with the plasmid mixture, proliferation of spleen cells (5000–8000 c.p.m.) was close to that shown by hamsters inoculated with 200 µg of pcDNA-Ehcp112 and much lower than that shown by animals inoculated with 100 µg of pcDNA-Ehadh112 (Fig. 5c).



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Fig. 5. Immune response of animals inoculated with the mixture of pcDNA-Ehadh112 and pcDNA-Ehcp112. Hamsters were immunized with 50 µg each of pcDNA-Ehcp112 and pcDNA-Ehadh112 and their immune response was evaluated. (a) Humoral response against E. histolytica determined by ELISA. The data correspond to the mean of the OD492 values from sera (1 : 500) of animals (n=8) immunized with the same dose and normalized with respect to the values obtained with the pre-immune sera at the same dilution. (b) Western blots using total E. histolytica proteins. Lanes: 1, sera from immunized animals; 2, anti-EhCPADH mAb; 3, pre-immune sera; 4, sera from animals immunized with pcDNA3. (c) [3H]Thymidine incorporation of lymphocytes obtained from the spleens of animals immunized under stimulation of a different number of E. histolytica trophozoites (open bars, 1x104; solid bars, 4x104). The data correspond to the mean c.p.m. values obtained from animals (n=8) immunized. Lines indicate SD.

 
Immune sera inhibit trophozoite adherence to human RBCs
Antibodies against the EhCPADH protein complex inhibit RBC adherence to the trophozoites (Garcia-Rivera et al., 1999). Here, we evaluated the effect of the antibodies independently generated against EhCP112 and EhADH112 and the mixture of both proteins on RBC adherence. We used a pool of immune sera (1 : 100) from hamsters independently immunized with pcDNA-Ehcp112 or pcDNA-Ehadh112, or with a mixture of both plasmids with similar titre. A significant adherence inhibition (P<0·01) was observed with the three immune sera (60–67 %) (Fig. 6, groups 3–5) with respect to the animals that received vector pcDNA3 (7 %) (Fig. 6, group 2). The adherence inhibition by the anti-EhCPADH mAb was 76 % (Fig. 6, group 6). The pre-immune sera inhibited trophozoite adherence by 23 % (Fig. 6, group 1).



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Fig. 6. Inhibition of E. histolytica trophozoite adherence by IgG from immunized animals. E. histolytica trophozoites were incubated for 20 min at 4 °C with (1) pre-immune sera or with sera of animals immunized with (2) pcDNA3, (3) pcDNA-Ehcp112, (4) pcDNA-Ehadh112 or (5) the mixture of both plasmids. Then, the adherence efficiency was determined. (6) Control using the anti-EhCPADH mAb. Zero per cent inhibition efficiency corresponds to values obtained in the absence of sera. The data represent the mean of three independent experiments performed in duplicate. Lines indicate SD.

 
Immunization with the plasmid mixture reduced hepatic damage
Next, we compared the effect of inoculation with either of the plasmids with inoculation with a mixture (50 µg of each) of them on the animals' sensitivity to liver abscess development. One week after the last DNA injection, following the described protocol, virulent trophozoites were intraportally inoculated into hamsters (n=8). One week later, the animals were anaesthetized and their livers were analysed carefully. Dramatic differences in liver damage were observed between the animals immunized with the plasmid mixture and the other groups (Fig. 7). All livers of the animals that were not immunized or were immunized with pcDNA3, pcDNA-Ehcp112 or pcDNA-Ehadh112 showed severe hepatic damage which covered almost all the organ (Fig. 7, c, v, cp, adh). These animals also showed hepatomegaly and multiple adherences to diaphragm and epiplon and distended colon. On the contrary, livers of hamsters immunized with the mixture of pcDNA-Ehcp112 and pcDNA-Ehadh112 presented a significant reduction in damage, abscesses were fewer and smaller, and the liver size was conserved (Fig. 7, cp/adh). Some abscesses were located in the opposite part of the portal system. Fig. 7 shows livers representing the different groups. Pictures show the larger abscesses found, independent of the side of the liver on which they appeared. These results indicate that in these conditions both EhCP112 and EhADH112 are required to produce a protective immune response.



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Fig. 7. Hepatic damage in immunized animals produced by live trophozoites. Liver abscesses detected 7 days after the challenge in animals (c) not immunized or immunized with (v) 100 µg of pcDNA3, (cp) 100 µg of pcDNA-Ehcp112, (adh) 100 µg of pcDNA-Ehadh112 or (cp/adh) 50 µg of each plasmid. (hl) Healthy liver of an animal that was not challenged.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this work, we studied recombinant EhCP112 and EhADH112 polypeptides expressed in transfected fibroblasts and animals. We used the Ehcp112 and Ehadh112 genes that encode for the EhCPADH protein complex (Garcia-Rivera et al., 1999). The full-length Ehcp112 gene, which encodes EhCP112, was used in these experiments to provoke its expression as a pre-proenzyme in mammalian cells and avoid the cell damage by the mature protein. However, the damage exhibited by Ehcp112-L929 cells may be due to the expression and processing of the cysteine protease inside the fibroblasts. Protease processing could be done through similar cellular mechanisms in trophozoites and fibroblasts. Only those cells able to resist the effect of EhCP112 survived and grew well, giving rise to a stable cell line that was resistant to the effect of this cysteine protease.

According to the TMPRED program (Hofmann & Stoffel, 1993), the polypeptides encoded by the Ehcp112 gene and the Ehadh112 gene fragment used here have transmembrane sequences. This allowed their expression in the fibroblast plasma membrane, in a way that was analogous to native protein expression in the trophozoites. The recombinant antigens may also be exposed on the hamster cells' surface favouring the stimulation of the humoral immune response.

EhCP112 has a signal peptide, explaining its presence in the Ehcp112-L929 cells. Intriguingly, EhADH112 does not have a detectable signal peptide, which poses the question as to how this recombinant protein went to the plasma membrane in the Ehadh112-L929 cells. One possibility is that its delivery to the plasma membrane could have been achieved by a signal-peptide-independent pathway similar to other membrane or secreted proteins reported in other organisms (Cho & Cummings, 1995; Denny et al., 2000; Gray et al., 1989; Kim et al., 1998; Lopez-Ordonez et al., 2001; Rubartelli et al., 1990; Trudel et al., 2000; Zhao & Morales, 2000). If this protein has a signal that allows it go to the plasma membrane, this should be situated at its C terminus, because the recombinant polypeptide used here does not contain the first 106 aa of the N terminus. There are many reports indicating that in some proteins the amino acid sequence required for membrane driving or for secretion is situated at the C terminus (Gray et al., 1989; Kim et al., 1998; Lopez-Ordonez et al., 2001). However, more studies are needed to confirm this.

The EhCPADH complex induces a good humoral response both in hamsters and humans (Arroyo & Orozco, 1987; Martinez-Lopez et al., 2004). The humoral response may account for the protection observed in the animals inoculated with the mixture of the plasmids. In fact, studies using both active and passive immunization strategies suggest that certain antibodies to E. histolytica may protect against liver abscesses (Lotter et al., 1997; Seydel et al., 1996; Zhang et al., 1994). The cellular response did not augment in the protected animals, although with these experiments we cannot discard differences in the quality of the cellular response with the different immunization protocols. Humoral response produced by the independent polypeptides and by the combination of them was rather low (Figs 3a and 5a), but specific, as was shown by the recognition of the EhCPADH protein complex by the hamster antibodies (Figs 3c, d and 5b). The low response could be due to the low amount of antigen produced by the plasmids. In ELISA assays we used heat-killed trophozoites as antigen; perhaps a better response would be obtained using the recombinant EhCP112 and EhADH112 proteins. However, recombinant EhCP112 protein was obtained in a very low amount, probably due to its toxicity, and both recombinant polypeptides are easily degraded.

The specificity of hamsters' antibodies was confirmed by their recognition of the EhCPADH complex in Western blot assays using total trophozoite proteins and by their adherence-inhibition ability in the in vitro assays. However, the in vivo virulence of the trophozoites was not affected in hamsters independently inoculated with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112. Our results and those of many others indicate that the complexity of in vivo virulence cannot be compared with in vitro studies.

Zhang & Stanley (1999) did not find differences in the antibody titres of vaccinated animals that were or were not protected from liver abscess. Here, we found that inoculation with both plasmids together improved the humoral response of the hamsters; however, we do not know the precise contribution of the specific antibodies to the reduction of liver damage observed in this group.

It has been reported that DNA vaccines promote the cellular response more than the humoral one (Kowalczyk & Ertl, 1999), and experimental evidence suggests that the cellular response has a predominant role in protection against amoebiasis (Campbell & Chadee, 1997; Schain et al., 1995), although the role of the humoral response has also been documented (Lotter et al., 1997; Seydel et al., 1996; Zhang et al., 1994).

Interestingly, inoculation with the plasmid mixture improved the immune response of the hamsters and reduced both the number and the size of the hepatic abscesses observed. It is conceivable that the expression in the animals of both polypeptides may promote the formation of a protein complex similar to the native EhCPADH in trophozoites. This could favour the presentation of more representative antigenic epitopes of the native complex to the immune system, providing a more-specific stimulation for T and B lymphocytes. However, this needs to be investigated further.


   ACKNOWLEDGEMENTS
 
This work was supported by the European Community. It was also supported by the Howard Hughes Medical Institute (USA) and CONACyT (Mexico). We are also grateful for the excellent technical assistance of Mr Alfredo Padilla-Barberi for the photographic work and we thank Dr Ortiz-Navarrete, CINVESTAV, IPN, Mexico, who kindly provided the L929 fibroblast cells.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Arroyo, R. & Orozco, E. (1987). Localization and identification of an Entamoeba histolytica adhesin. Mol Biochem Parasitol 23, 151–158.[CrossRef][Medline]

Campbell, D. & Chadee, K. (1997). Interleukin (IL)-2, IL-4, and tumor necrosis factor-alpha responses during Entamoeba histolytica liver abscess development in gerbils. J Infect Dis 175, 1176–1183.[Medline]

Cho, M. & Cummings, R. D. (1995). Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. II. Localization and biosynthesis. J Biol Chem 270, 5207–5212.[Abstract/Free Full Text]

Denny, P. W., Gokool, S., Russell, D. G., Field, M. C. & Smith, D. F. (2000). Acylation-dependent protein export in Leishmania. J Biol Chem 275, 11017–11025.[Abstract/Free Full Text]

Diamond, L. S., Harlow, D. R. & Cunnick, C. C. (1978). A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72, 431–432.[Medline]

Engvall, E. & Perlman, P. (1971). Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874.[CrossRef][Medline]

Garcia-Rivera, G., Sanchez, T., Orozco, E. & Guarneros, G. (1982). Isolation of clones of E. histolytica deficient in adhesion to human erythrocytes. Arch Invest Med (Mex) 13 Suppl 3, 129–136 (in Spanish).[Medline]

Garcia-Rivera, G., Rodriguez, M. A., Ocadiz, R., Martinez-Lopez, M. C., Arroyo, R., Gonzalez-Robles, A. & Orozco, E. (1999). Entamoeba histolytica: a novel cysteine protease and an adhesin form the 112 kDa surface protein. Mol Microbiol 33, 556–568.[CrossRef][Medline]

Gaucher, D. & Chadee, K. (2002). Construction and immunogenicity of a codon-optimized Entamoeba histolytica Gal-lectin-based DNA vaccine. Vaccine 20, 3244–3253.[CrossRef][Medline]

Ghadirian, E., Meerovitch, E. & Hartmann, D. P. (1980). Protection against amebic liver abscess in hamsters by means of immunization with amebic antigen and some of its fractions. Am J Trop Med Hyg 29, 779–784.[Medline]

Gray, L., Baker, K., Kenny, B., Mackman, N., Haigh, R. & Holland, I. B. (1989). A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. J Cell Sci Suppl 11, 45–57.[Medline]

Hofmann, K. & Stoffel, W. (1993). TMbase – A database of membrane spanning proteins segments. Biol Chem Hoppe Seyler 374, 166.

Kim, K. S., Bae, K. H., Kim, I. C., Byun, S. M. & Shin, Y. C. (1998). Streptokinase secretion by Serratia marcescens signaled by the C-terminal 41 amino acid segment of metalloprotease. Biochem Mol Biol Int 45, 725–733.[Medline]

Kowalczyk, D. W. & Ertl, H. C. (1999). Immune responses to DNA vaccines. Cell Mol Life Sci 55, 751–770.[CrossRef][Medline]

Lopez-Ordonez, T., Rodriguez, M. H. & Hernandez-Hernandez, F. D. (2001). Characterization of a cDNA encoding a cathepsin L-like protein of Rhodnius prolixus. Insect Mol Biol 10, 505–511.[CrossRef][Medline]

Lotter, H., Zhang, T., Seydel, K. B., Stanley, S. L., Jr & Tannich, E. (1997). Identification of an epitope on the Entamoeba histolytica 170-kD lectin conferring antibody-mediated protection against invasive amebiasis. J Exp Med 185, 1793–1801.[Abstract/Free Full Text]

Martinez-Lopez, C., Orozco, E., Sanchez, T., Garcia-Perez, R. M., Hernandez-Hernandez, F. & Rodriguez, M. A. (2004). The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites. Cell Microbiol 6, 367–376.[Medline]

Petri, W. A., Jr, Smith, R. D., Schlesinger, P. H., Murphy, C. F. & Ravdin, J. I. (1987). Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica. J Clin Invest 80, 1238–1244.[Medline]

Rodriguez, M. A., Hernandez, F., Santos, L., Valdez, A. & Orozco, E. (1989). Entamoeba histolytica: molecules involved in the target cell–parasite relationship. Mol Biochem Parasitol 37, 87–99.[CrossRef][Medline]

Rubartelli, A., Cozzolino, F., Talio, M. & Sitia, R. (1990). A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J 9, 1503–1510.[Abstract]

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463–5467.[Abstract]

Schain, D. C., Salata, R. A. & Ravdin, J. I. (1995). Development of amebicidal cell-mediated immunity in gerbils (Meriones unguiculatus) immunized with the galactose-inhibitable adherence lectin of Entamoeba histolytica. J Parasitol 81, 563–568.[Medline]

Seydel, K. B., Braun, K. L., Zhang, T., Jackson, T. F. & Stanley, S. L., Jr (1996). Protection against amebic liver abscess formation in the severe combined immunodeficient mouse by human anti-amebic antibodies. Am J Trop Med Hyg 55, 330–332.[Medline]

Soong, C. J., Kain, K. C., Abd-Alla, M., Jackson, T. F. & Ravdin, J. I. (1995a). A recombinant cysteine-rich section of the Entamoeba histolytica galactose-inhibitable lectin is efficacious as a subunit vaccine in the gerbil model of amebic liver abscess. J Infect Dis 171, 645–651.[Medline]

Soong, C. J., Torian, B. E., Abd-Alla, M. D., Jackson, T. F., Gatharim, V. & Ravdin, J. I. (1995b). Protection of gerbils from amebic liver abscess by immunization with recombinant Entamoeba histolytica 29-kilodalton antigen. Infect Immun 63, 472–477.[Abstract]

Stanley, S. L., Jr, Becker, A., Kunz-Jenkins, C., Foster, L. & Li, E. (1990). Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats. Proc Natl Acad Sci U S A 87, 4976–4980.[Abstract]

Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.[Abstract]

Trudel, C., Faure-Desire, V., Florkiewicz, R. Z. & Baird, A. (2000). Translocation of FGF2 to the cell surface without release into conditioned media. J Cell Physiol 185, 260–268.[CrossRef][Medline]

Tsutsumi, V., Mena-Lopez, R., Anaya-Velazquez, F. & Martinez-Palomo, A. (1984). Cellular bases of experimental amebic liver abscess formation. Am J Pathol 117, 81–91.[Abstract]

Vinayak, V. K., Sawhney, S., Jain, P. & Chakravarti, R. N. (1980). Protective effects of crude and chromatographic fractions of axenic Entamoeba histolytica in guinea-pigs. Trans R Soc Trop Med Hyg 74, 483–487.[CrossRef][Medline]

Vinayak, V. K., Purnima & Saxena, A. (1987). Immunoprotective behaviour of plasma-membrane-associated antigens of axenic Entamoeba histolytica. J Med Microbiol 24, 297–302.[Abstract]

Walsh, J. A. (1986). Problems in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Rev Infect Dis 8, 228–238.[Medline]

Zhang, T. & Stanley, S. L., Jr (1996). Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess. Infect Immun 64, 1526–1531.[Abstract]

Zhang, T. & Stanley, S. L., Jr (1999). DNA vaccination with the serine rich Entamoeba histolytica protein (SREHP) prevents amebic liver abscess in rodent models of disease. Vaccine 18, 868–874.[CrossRef][Medline]

Zhang, T., Cieslak, P. R., Foster, L., Kunz-Jenkins, C. & Stanley, S. L., Jr (1994). Antibodies to the serine rich Entamoeba histolytica protein (SREHP) prevent amoebic liver abscess in severe combined immunodeficient (SCID) mice. Parasite Immunol 16, 225–230.[Medline]

Zhao, Q. & Morales, C. R. (2000). Identification of a novel sequence involved in lysosomal sorting of the sphingolipid activator protein prosaposin. J Biol Chem 275, 24829–24839.[Abstract/Free Full Text]

Received 25 November 2003; revised 10 February 2004; accepted 13 February 2004.



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