Parasitol Res (2011) 108:439–449 DOI 10.1007/s00436-010-2085-6
ORIGINAL PAPER
Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses Enrique González & Maria del Carmen García de Leon & Isaura Meza & Rodolfo Ocadiz-Delgado & Patricio Gariglio & Angelica Silva-Olivares & Silvia Galindo-Gómez & Mineko Shibayama & Patricia Morán & Alicia Valadez & Angelica Limón & Liliana Rojas & Eric G. Hernández & René Cerritos & Cecilia Ximenez
Received: 30 June 2010 / Accepted: 8 September 2010 / Published online: 5 October 2010 # Springer-Verlag 2010
Abstract Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90–100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic E. González : M. d. C. García de Leon : P. Morán : A. Valadez : A. Limón : L. Rojas : E. G. Hernández : R. Cerritos : C. Ximenez (*) Depto. de Medicina Experimental, Facultad de Medicina, UNAM, Dr. Balmis No 148, Col. Doctores, CP 06726 México, D.F., Mexico e-mail:
[email protected]
inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1–2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host–parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.
Introduction I. Meza Depto. de Biomedicina Molecular, CINVESTAV, Ave. Politécnico Nacional No 2508, CP 07360 México, D.F., Mexico R. Ocadiz-Delgado : P. Gariglio Depto. de Genética y Biología Molecular, CINVESTAV, Ave. Politécnico Nacional No 2508, CP 07360 México, D.F., Mexico A. Silva-Olivares : S. Galindo-Gómez : M. Shibayama Depto. de Infectómica y Patogénesis Molecular, CINVESTAV, Ave. Politécnico Nacional No 2508, CP 07360 México, D.F., Mexico
Calreticulin (CRT) is a calcium (Ca2+)-binding protein mostly located in the endoplasmic reticulum (ER). It is a highly conserved multifunctional protein found across a diverse range of species (Michalak et al. 1999). The CRT is one of the immunogenic molecules of Entamoeba histolytica that induces an antibody response in the human host. Previously, our group performed an antigenic recognition analysis on serum samples from amoebic dysentery or amoebic liver abscess (ALA) patients. The analysis showed that 95% of dysentery patients and 100% of ALA patients
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recognized a 51-kDa fraction of E. histolytica HM1:IMSS that, once partially sequenced, was shown to be a CRT-like protein (González et al. 2002). Recently, the sequence of the E. histolytica HM1:IMSS genome was reported, and two putative Ehcrt genes, one of 1,178 bp (GB-EAL649855.1) and one of 417 bp (gi: 67466599), were reported (Loftus et al. 2005). Based on these data, we cloned and expressed the Ehcrt gene. The Ehcrt gene is located in the nuclear DNA of this parasite, from which the entire sequence of the 1,178-bp fragment was obtained. The size of the sequence correlates with the predicted molecular weight of the E. histolytica CRT-like protein reported previously (González et al. 2002). The proteomic analysis of phagosomes isolated from E. histolytica using MALDI-TOF mass spectrometry demonstrated the presence of CRT in E. histolytica HM1:IMSS strain (Okada et al. 2005). The CRT-like protein reported in our previous work (González et al. 2002) shows similar molecular weight with the one reported by Tolstrup et al. (2007). On the other hand, the exact location of CRT in both E. histolytica and Entamoeba dispar and its biological functions are unknown. However, there is recent evidence that supports the previous findings that suggest a subcellular location of the protein in small vesicles of heterogeneous sizes that show no continuity or reticular organization (Ghosh et al. 1999; Girard-Misguich et al. 2008). In the present work, we show for the first time the expression of the Ehcrt gene and the immunohistochemical detection of CRT in trophozoites of tissue lesions of amoebic liver abscesses induced in a hamster model. We use specific antibodies against the recombinant protein EhCRT. Nevertheless, the importance of CRT in the physiology of E. histolytica and its role in the pathological capabilities of this parasite remain to be defined.
Material and methods Expression of EhCRT recombinant proteins The amplification of the Ehcrt gene was performed with specific oligonucleotides and nuclear genomic DNA of E. histolytica. The cloning and expression of recombinant CRT proteins were performed as described previously (Holton and Graham 1991). Briefly, the plasmid bluescrib-KS + (pBSKS+) was used for cloning of PCR products. We obtained three clones named pb-EhCRT-1200, pb-EhCRT-N, and pb-EhCRT-C, which were verified through partial sequencing, all corresponding to sequences of nucleotides of the crt gene (GenBank accession number GU477560). The mentioned clones correspond to the complete gene and to each half of the crt gene of E. histolytica, respectively.
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These recombinant plasmids were sub-cloned into the prokaryotic expression vector pProEX HT–b™ (Life Technologies, Grand Island, NY, USA) coupled to a sixhistidine tag on the NH2 end. The cloning was directed with the restriction sites BamH1/Xho1. Escherichia coli BL21 competent bacteria were transformed with the respective recombinant plasmid. The expression of recombinant protein EhCRT-1200 and EhCRT-C was induced with a final concentration of 1 mM IPTG. The cells were harvested by centrifugation at 3,000×g for 12 min, and the bacterial pellet was re-suspended in 5 ml of the lysis buffer (8 M urea, 0.1 M NaH2PO4, and 0.1 M Tris–HCl; pH 8.0). The lysate was added to a 50% suspension of Ni-NTA agarose (Qiagen; Valencia, CA, USA) and incubated at room temperature for 1 h with agitation. The mixture was filtered through a filtration column (Qiagen; Valencia, CA, USA), and the recombinant proteins were eluted with 8 M urea buffer, pH 4.5, under denaturing conditions. For expression and purification of recombinant proteins, the Qiagen kit QIAexpressionist system was used (Qiagen; Valencia, CA, USA). The selected fractions were dialysed against 19 mM phosphate-buffered saline (PBS) to eliminate the urea and then submitted to electrophoresis in 10% acrylamide gels. This procedure allowed us to obtain an 1,178-bp PCR product that corresponded to the complete Ehcrt gene and encoded the complete protein (EhCRT-1200) and one small amplification product of 650 bp that encoded the Cterminal end of EhCRT. Production of specific antibodies against recombinant EhCRT For antibody production, 9-week-old BALB/c mice were bled 1 week prior to immunization to obtain pre-immune serum, were then immunized subcutaneously with 50 μg of EhCRT-1200 or EhCRT-C suspended in 0.1 ml of PBS solution and emulsified in the same volume of Freund’s complete adjuvant (Sigma Chemical Co. St Louis, MO, USA). The mice received three additional intradermal immunizations with 50 μg of the proper recombinant protein suspended in 0.1 ml PBS 15 days after the first immunization. Doses were administered in 15-day intervals. The immune serum was collected 10 days after the last immunization and thereafter precipitated with an ammonium sulfate-saturated solution to obtain the gamma-globulin fraction. The specific anti-EhCRT-1200 and EhCRT-C IgG antibodies were obtained by affinity chromatography on a Sepharose 4B coupled with protein A column (Sigma Chemical Co. St Louis, MO, USA). The purity of IgG was tested by electrophoresis in 10% acrylamide gels stained with Coomassie blue. The specificity was tested by Western
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blot using the E. histolytica membrane-rich extract as previously described (Ximénez et al. 1993) or respective recombinant EhCRT as antigens. For the western blot analysis (Towbin et al. 1979), 10 μg of the membrane-rich extract or recombinant protein were transferred to nitrocellulose paper, and reacted with affinity purified IgG anti-E. histolytica HM1:IMSS and affinity purified IgG against recombinant proteins diluted 1:250, using as secondary antibody goat anti-mouse IgG coupled to alkaline phosphatase and the reaction was developed with the substrate (NBT/BCIP; Sigma Chemical Co. St Louis, MO, USA). Confocal assay Detection of EhCRT was performed by confocal microscopy. Trophozoites were grown under axenic conditions using TYI-S33 culture medium for 48 h as previously described (Diamod et al. 1978). After incubation, trophozoites were detached and then cultured in Petri dishes containing cover slips in the presence of TYI-S33 medium at 37°C for 4 h. The culture medium was removed and the cover slips were washed in pre-warmed PBS to avoid amoeba detachment. The fixed cells were permeabilized with 0.1% (v/v) Triton X100 and blocked with 3% bovine serum albumin (BSA; Sigma Chemical Co. St Louis, MO, USA). Then the trophozoites were incubated with the specific antibody described previously and diluted 1:50 in 1% PBS/BSA for 1 h at 37°C. Slides were washed several times with PBS solution and incubated for 1 h with a fluorescein-conjugated rabbit anti-mouse IgG–FITC (Zymed, San Francisco, CA, USA) diluted 1:500 in the same buffer. After that the slides were incubated for 10 min with propidium iodide (Pi). At the end of the incubation the slides were rinsed with PBS and mounted with 4 μl of vecta-shield anti-fade solution (Sigma Chemical Co., St Louis, MO, USA). For co-localization assays, cells were treated as described above for immunofluorescence but with some modifications. In this assay mixtures of IgGs were used as primary antibodies [mouse anti-EhCRT IgG and rabbit anti-Eh-protein disulfide isomerase (EhPDI) IgG; the last kindly provided by Dr. Marco Antonio Ramos, UABC–Mexico]. The PDI is a protein retained in the ER and has been used as a molecular marker for the ER in different organisms. Thereafter, a mixture of secondary antibodies was used to reveal the antigen–antibody reaction (rhodamine-conjugated goat anti-rabbit IgG and fluorescein-conjugated rabbit antimouse IgG diluted 1:500; Zymed; San Francisco, CA, USA). Samples were examined under a confocal microscope (DM1RE–2, Leica) using appropriate fluorescence emission filters. Image acquisition (z-series) was performed with image processing software (Leica, LCS Lite Profile Pro.) using 0.5-μm steps. The images correspond to the maximum intensity projection of the z-series.
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Experimental amoebic liver abscess and immunohistochemical staining of parasites E. histolytica trophozoites strain HM1:IMSS were recovered from experimentally induced acute amoebic liver abscesses in golden hamsters (Mesocricetus auratus) after 7 days of inoculation. The initial axenic culture was started with a density of 7×105 trophozoites in 50 ml of TYI-S33 medium (Diamod et al. 1978) in plastic bottles. After 48 h of incubation at 36.5°C, trophozoites were harvested, washed twice in PBS, and their viability was determined by the trypan blue exclusion technique. Experimental acute ALA was produced in 100-g hamsters following a technique described by Olivos-Garcia et al. (2004). Briefly, 2.5×105 axenic trophozoites of E. histolytica were inoculated into the portal vein of anesthetized hamsters. After 5, 15, and 30 min; 1, 3, 6, and 12 h; and 1, 2, and 5 days, animals (five hamsters for time) were sacrificed by an ether overdose and the liver was removed and fixed for 48 h in 4% paraformaldehyde in PBS, followed by dehydration and paraffin embedding. Serial sections of 6 μm thick were obtained and de-paraffinized from tissue blocks, lesions were identified by hematoxylin/eosin stain. Samples selectioned were blocked with 3% PBS/BSA solution, reacted with specific mouse anti-EhCRT antibody diluted 1:25, and incubated at 4°C overnight. Antigen−antibody reaction was detected using goat anti-mouse IgG antibody coupled to alkaline phosphatase (Zymed Laboratories, San Francisco, CA, USA) diluted 1:100; NBT/BCIP substrate (Roche Diagnostics GmbH; Mannheim, Germany) was used as the chromogen. Monoclonal mouse IgG1 antibody against Aspergillus niger glucose oxidase was used as the negative control (clone DAK-GO1, code # X09931; Dako; Golstrup, Denmark); the positive control was a mouse polyclonal IgG against E. histolytica membrane-rich extract described previously (Ximénez, et al. 1993). To avoid cross-reaction with CRT from hamster hepatic tissue anti-EhCRT antibodies were adsorbed with a lyophilized extract of hamster liver. The samples were counterstained with aqueous eosin (García de Leon et al. 2006). The protocols for animal care were previously approved by the Institutional Committee. The institution fulfills all the technical specifications for the production, care, and use of laboratory animals and is certified by a National Law (NOM-062-ZOO-1999). All hamsters were handled according to the guidelines of the 2000 AVMA Panel of Euthanasia. In situ RT-PCR The detection of EhCRT mRNA was carried out using a two-step in situ RT-PCR procedure as previously reported with some modifications (Nuovo 1996, 2001, 2006, 2007,
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Martinez et al. 1995; Ocadiz-Delgado et al. 2008, 2009). Tissue sections of hamsters’ liver previously selected (three sections each time after intraportal inoculation) were pretreated with 0.5 μg/μl proteinase K (Sigma Aldrich, St Louis, MO, USA) in 0.01 M PBS, pH 7.4, at room temperature for 30 min. After proteinase K digestion, tissues were treated with 1 U/sample of DNase I, RNasefree (Roche Diagnostics GmbH, Mannheim, Germany) for 48 h at room temperature. After washing with DEPC-treated water, reverse transcription was performed using SuperScript II reverse transcriptase following the manufacturer’s specifications (Invitrogen, Carlsbad, CA, USA). Briefly, 25 μl DEPC-treated water containing 2.5 μl oligo (dT), 10 mM dNTP mix (10 mM each dATP, dGTP, dCTP, and dTTP at neutral pH), 5 μl 5× first-strand buffer, 1.2 μl 0.1 M DTT, 0.25 μl recombinant ribonuclease inhibitor (40 U/μl), and reverse transcriptase (100 U/section; Invitrogen, Carlsbad, CA, USA) were added to each section. Slides were incubated at 42°C for 2 h in a sealed humidified chamber. After washes, direct in situ PCR was performed using the specific primers described in the next section. As a positive control, detection of Ehβ-actin constitutive housekeeping gene expression was used. In addition, a negative control was included substituting one of the primers by H2O in the PCR reaction. Synthesized cDNAs in the in situ RT assays were recovered, and thereafter used for Real-time PCR. Amplification of Ehcrt gene by in situ PCR Direct in situ PCR was performed as previously described, with modifications (Nuovo 1996, 2001, 2006, 2007; Martinez et al. 1995; Ocadiz-Delgado et al. 2008, 2009). The tissue sections after washing with ultrapure water, 50 μl of the PCR master mix solution containing digoxigenin-11-(2′-deoxy-uridine-5′)-triphosphate (DIG11-Dutp; Roche Diagnostic GmbH, Mannheim, Germany) were added. To reduce primer dimer formation, the PCR solution was heated to 70°C for 10 min before Taq DNA polymerase (5 U per reaction) was added. Negative controls were made without primers or Taq. In situ PCR was performed using the system provided by Perkin Elmer (Perkin Elmer/ Applied Biosystems, Carlsbad, CA, USA). The slides were preheated to 70°C on the assembly tool included in the in situ Perkin Elmer equipment, 50 μl of PCR master mix were added to each sample, and the reaction was sealed using AmpliCover disks and clips (Perkin Elmer/Applied Biosystems; Carlsbad, CA, USA). After assembly, slides were placed at 70°C in the GeneAmp in situ PCR system 1,000 (Perkin Elmer/Applied Biosystems, Carlsbad, CA, USA) until use. PCR amplification was performed for 18 cycles with the pre-validated Ehcrt5′ and Ehcrt3′ specific primers. These
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primers amplify the C-end region of the Ehcrt gene: sense, Ehcrt–F (5′ GGA TCC CAA GAA GGA AAG TTT GAT GAA 3′) and antisense, Ehcrt–R (5′ CTC GAG TTA AAG CTC TTC TTT GTT TTC 3′). As the internal constitutive expression control, Ehβ-actin transcripts were detected using the following primers: Ehβ-actin (sense), 5′ AGC TGT TCT TTC ATT ATA TGC 3′ and Ehβ-actin (antisense), 5′ TTC TCT TTC AGC AGT AGT GGT 3′, these primers amplified an insert of 180 bp. Reactions were performed in a 50-μl reaction using a GeneAmp in situ PCR 1,000 system (Perkin Elmer/ Applied Biosystems, Carlsbad, CA, USA). Reaction mixtures contained 0.2 mM of each primer, and amplifications were performed using the following solutions: 0.2 mM each deoxynucleotide triphosphate (dATP, dGTP, and dCTP), 0.19 mM dTT, 0.01 mM digoxigenin-11dUTP, and 20 mM Tris–HCl, pH 7.4 (50 mM KCl, 1.5 mM MgCl 2 ). We used recombinant Taq DNA polymerase (10 U/reaction; Invitrogen; Carlsbad, CA, USA). For amplification, cycles comprised a 1-min denaturing step at 94°C, a 1-min annealing step at 58°C, and a 1-min elongation step at 72°C. Then, a final cycle comprising a 5-min at 72°C was performed (Martinez et al. 1995; Nuovo 1996, 2001, 2006, 2007, Livak and Schmittgen 2001; Manjarrez et al. 2006). After cycling was complete, the temperature was kept at 4°C until disassembly. Clips were removed, AmpliCover disks were very carefully lifted from the slides without moving them sideways, and slides were washed for 5 min in PBS followed by 5 min in 100% EtOH before they were airdried. To ensure consistency and reproducibility and to eliminate PCR artifacts, all assays were performed in duplicate at a minimum of three separate occasions. Detection of in situ PCR products An indirect immunolabeling method using a primary anti-digoxigenin antibody (Fab fragments) (Roche Diagnostics GmbH; Mannheim, Germany) conjugated to alkaline phosphatase was chosen to detect the PCR product. Blocking was carried out in 5% BSA in PBS for 30 min. Slides were then drained, and an anti-DIG antibody diluted 1:200 in 100 mM Tris–HCl, pH 7.4, and 150 mM NaCl was applied (100 μl per sample) for 2 h at room temperature. As a negative control, the primary antibody was omitted. Detection of alkaline phosphatase was carried out for approximately 20 min using an NBT/BCIP kit (Zymed, San Francisco, CA, USA). After detection, all slides were simultaneously rinsed in distilled water for 5 min to stop the reaction and air-dried before mounting in Permount histological mounting medium (Fisher Scientific Ltd, Leicestershire, UK).
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Digital image capture, analysis, and quantification All photomicrographs were obtained using a Hyper HAD color video camera (Model SSC-DC30; Sony Corporation, Japan). The following method of semi-quantification was used for in situ RT-PCR analysis. After acquisition of the images using the digital camera, the experimental image files were opened in PhotoImpact software (Ulead PhotoImpact SE version 3.02; Ulead Systems, USA). The images were digitally processed to obtain better and more homogeneous signals and then selected for analysis of relevant regions. The selected regions were then digitally analyzed using ImageProPlus analysis software (Media Cybernetics, Inc., USA). Relative mRNA quantification by real-time PCR The relative quantification of the investigated samples by real-time PCR was performed using the previously synthesized cDNA in the in situ RT assays. For this purpose a 7300 Applied Biosystems apparatus (Applied Biosystems, Carlsbad, CA, USA) and the Quantitec SYBR green PCR kit were used (Qiagen, Valencia, CA, USA). PCR was performed for 60 cycles of a three-step PCR, including 10 s of denaturation at 95°C, a 30-s primer-dependent annealing phase at 57°C, and a 10-s template-dependent elongation at 72°C. The amplification of each template was performed in duplicate in one PCR run. The differential expression of the investigated genes was calculated as the ratios normalized to Ehβ-actin.
Fig. 1 Western blot of E. histolytica membrane-rich extract and recombinant proteins reacted with purified IgGs of hyper-immune serum anti-EhCRT recombinant proteins. Lanes 1–5 10 μg of E. histolytica membrane-rich extract was transferred to nitrocellulose sheets and reacted against: (1) mouse IgG anti-Eh antigen (positive control); (2) pre-immune mouse serum IgG (negative control); (3) second antibody control (goat anti-mouse IgG-AP); (4) anti-EhCRT-1200 IgG; and (5) anti-CRT-C IgG. Lanes 6–7 corresponds to nitrocellulose paper transferred with EhCRT1200, lanes 8–9 with EhCRT-C. Lane 6 was reacted against anti-EhCRT-1200 IgG; lane 7 reacted with anti-CRT-C IgG; lane 8 reacted with anti-EhCRT-1200 IgG; and lane 9 reacted with anti- EhCRT-C IgG. Standard molecular weights are indicated
Confocal microscopy detection of EhCRT The immunolocalization of the EhCRT protein is shown in Fig. 2. The distribution of the fluorescent signal of the anti-
Data analysis using 2-ΔΔCT Real-time PCR was performed on the corresponding cDNA synthesized from each sample. The data were analyzed using the equation described by Livak and Schmittgen (2001) as follows: amount of target=2-ΔΔCT. The threshold cycle (CT) indicates the fractional number at which the amount of amplified target reaches a fixed threshold, ΔCT = (average CRT CT−average actin CT), ΔΔCT = (average ΔCT at different times−average ΔCT 5 min after infection time) (calibrator). Validation of the method was performed as previously reported (Yalcin 2004).
Results Western blot analysis Figure 1 shows the Western blot of recombinant proteins reacted with mouse anti-EhCRT IgG antibodies. These antibodies recognize the recombinant proteins EhCRT-1200 and EhCRT-C, and also the 51-kDa fraction of the membrane-rich extract of E. histolytica HM1:IMSS strain.
Fig. 2 Detection of EhCRT in E. histolytica trophozoites by confocal microscopy analysis. Trophozoites were fixed, permeabilized, and incubated with specific antibodies: a trophozoite nucleus stained with propidium iodide; b trophozoite reacted with mouse anti-EhCRT1200 IgG; c merge of Pi-stained trophozoites nucleus and detection of EhCRT; and d represent the differential interference contrast. FITC-conjugated anti-mouse IgG was used as a secondary antibody. The micrographs show the maximal projection of the z-series. Scale bar represents 20 μm
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recombinant EhCRT antibody is mainly observed in different size cytoplasmic vesicles mostly distributed near the surface membrane as well as around the nucleus. However, heterogeneity on the amount of fluorescent mark displayed by trophozoites is also evident. This heterogeneity Fig. 3 Co-localization of EhCRT with the ER resident protein EhPDI analyzed by confocal microscopy. a and b Represent different patterns of immunodetection. a rabbit anti-EhPDI and with anti-rabbit IgG-TRIC; b trophozoites reacted with mouse antiEhCRT1200 and FITCconjugated secondary antibody; c co-localization of EhCRT with the ER resident protein EhPDI (chanel mergin); and d represent the differential interference contrast. The micrographs show the maximal projection of the z-series, and the cellular localization of both proteins in cytoplasmic vesicles distributed near to the surface membrane. Scale bar represents 20 μm
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may be due to differences in the stage of cell cycle in trophozoites since our culture is not synchronic. On the other hand, the co-localization assays show that EhCRT distribution is coincident with the one observed of the ER resident protein EhPDI (Fig. 3).
Parasitol Res (2011) 108:439–449 Fig. 4 Representative images of immunohistochemical detection of EhCRT in tissue sections of livers from hamsters inoculated with HM1:IMSS trophozoites via the portal vein and sacrificed at different stages after inoculation. a–c Correspond to control slides: tissue section taken 30 min after trophozoites inoculation stained with: a the hematoxylin/eosin stain; b tissue section treated with the negative antibody previously described, and with mouse IgG against E. histolytica HM1:IMSS membrane-rich extract (c). The next section d–j correspond to tissue sections treated with mouse IgG against EhCRT1200; d 30 min, e 1 h, f 6 h, g 12 h, h 1 day, i 2 days, and j 5 days, after intraportal trophozoites inoculation. The arrows point out some demonstrative trophozoites. Scale bar represents 20 μm
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Immunochemical detection of EhCRT in amoebic liver abscess lesions Representative sections of hepatic tissue obtained at different times after the intraportal inoculation of E. histolytica HM1:IMSS virulent trophozoites (30 min; 1, 6, and 12 h; and 1, 2, and 5 days) are shown in Fig. 4. The immunodetection of EhCRT in the trophozoites established in the hepatic tissue is evident and displays a similar distribution on trophozoites as observed in the confocal microscopy assays (Fig. 2). The immunocytochemical signal is displayed in different-size cytoplasm vesicles. In some sections these vesicles are mainly located near the cell surface membrane (Figs. 2 and 4). Moreover, E. histolytica trophozoites apparently do not secrete or export the EhCRT protein into the hepatic tissue. In situ RT-PCR The estimation of expression of the Ehcrt gene in trophozoites into hepatic lesions of ALA was performed at different times during the development of the abscess; the amplification of the EhCRT mRNA was detected using an anti-digoxigenin antibody. The amplification was performed with oligonucleotides specific for Ehcrt and Ehβ-actin as a positive control; representative microphotographs of the assays were taken (data not shown). The presence of EhCRT mRNA was evaluated through densitometric analysis of in situ RT-PCR. The values are shown in Fig. 5a where a significant increase in the expression of the Ehcrt gene can be observed, particularly 30 min after intrahepatic inoculation of E. histolytica trophozoites. The highest relative signal was observed at 60 min; however, it is clear that the expression decreased abruptly between 2 and 6 h after inoculation. These findings correlate with the microscopic detection of EhCRT cDNA (data not shown). Real-time RT-PCR The relative quantification of EhCRT mRNA expression is shown in Fig. 5b; the values correspond to the relative expression of cDNA for EhCRT into ALA specimens assayed by real-time RT-PCR. Values of relative expression clearly increased at 30 min after intraportal trophozoite inoculation, reaching a peak after 60 min; thereafter, the expression fell sharply to low levels. These values are in agreement with those obtained from the densitometric analysis shown in Fig. 5a.
Discussion E. histolytica displays particular biological characteristics, for example, the absence of mitochondria and peroxisomes,
Fig. 5 In situ RT-PCR to determine the expression of EhCRT in trophozoites present in tissue sections of livers from hamsters inoculated with E. histolytica HM1:IMSS. a Densitometric analysis of the in situ RT-PCR expression of the mRNA for the EhCRT. b Relative quantification of expression of the mRNA for EhCRT performed by real-time RT-PCR. Mean of three separate assays. *p< 0.005
a non-recognizable Golgi apparatus, and lack of structural ER. These features have been the source of different hypotheses concerning the lineage of E. histolytica to the eukaryotes organisms (El-Hashimi and Pittman 1970; Lowe and Maegraith 1970; Rosenbaum and Wittner 1970; Bakker-Grunwald, and Wostmann 1993; Clark and Roger 1995). Previous data on transmission electron microscopy and confocal microscopy of pre-fixed E. histolytica trophozoites suggest that the endoplasmic reticulum is a network of vesicles of heterogeneous sizes (Mazzuco et al. 1997; Ghosh et al. 1999). CRT is a protein that can be relocated into the cytoplasmic membrane of motile trophozoites, as was demonstrated by Girard-Misguich et al. (2008) through the induction of capping using Concanavalin A. In the present work, we produced both E. histolytica recombinant EhCRT and mono-specific anti-EhCRT antibodies that allowed for the immunodetection of CRT in virulent E. histolytica HM1:IMSS trophozoites. The distribution pattern of CRT detected in trophozoites both in vitro and in the liver tissues at different times after intraportal
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inoculation of trophozoites (Figs. 2 and 4) shows a cytoplasm dotted with a heterogeneous distribution in vesicular structures. As already mentioned, these images are highly similar to those described by Girard-Misguich et al. (2008). The association of EhCRT with the ER was simultaneously monitored with anti-EhPDI in the confocal microscopy assay. Both PDI and EhCRT co-localize on the same trophozoite structures, demonstrating that EhCRT is a protein linked to the ER. In contrast, we have to mention recent data obtained through ER target GFP-fusion protein and the fluorescence loss in photobleaching (FLIP) technique that unequivocally demonstrated the presence of a continuous endoplasmic reticulum compartment in living trophozoites (Teixeira and Huston 2008). These findings have important evolutionary implications. Other authors have mentioned that the presence of a continuous endoplasmic reticulum points to the divergence of this protozoan from other eukaryotic lineages relatively late in evolution. It is in these structures that a number of proteins associated with vesicular trafficking, such as small GTPases (Rab family proteins), BiP protein (Hsp70 protein family), PDI, ERD2, and calreticulin, are located (Sánchez-López et al. 1998; Manning-Cela et al. 2003; Okada et al. 2005; Girard-Misguich et al. 2008; Teixeira and Huston 2008). The immunocytochemical assays performed in the amoebic liver abscess induced in the hamster model suggest that trophozoites over-express the Ehcrt gene during the early stages (30 min–2 h) of development of ALA, followed by an abrupt decrease to the original level of expression. The period of over-expression may be the consequence of an adaptation process of the parasite to the new environment. This has been described in other parasite–host interactions, in particular, in those parasites with life cycles involving both vertebrate and invertebrate hosts (Joshi et al. 1996; Souto-Padron et al. 2004). In this context, the up-regulation of CRT mRNA from promastigote to amastigote forms has been demonstrated in Leishmania species, indicating that expression of CRT responds rapidly to environmental changes (Joshi et al. 1996; Nakashi et al. 1998). In our model, the intraportal inoculation of trophozoites could in fact be a kind of physiological stress that involves the adaptation to changes in temperature, pH, and/or exposure to host defenses. In contrast, with other parasite infection models (Rokeach et al. 1994; El-Gengehi et al. 2000), we did not detect EhCRT in the hepatic tissue. This may indicate that E. histolytica trophozoites do not secrete or export CRT into the liver parenchyma. As was mentioned before, E. histolytica CRT and other parasite CRTs are highly immunogenic in the human host. Recombinant hookworm CRT binds to and inhibits the biological function of human C1q; moreover, CRT binds specifically to the cytoplasm signaling domains of various
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integrins, which are important adhesion molecules in leukocyte and platelet functions interfering with the immune response against this parasite (Kasper et al. 2001). In E. histolytica, one of the various virulence factors is the well-studied complement resistance of virulent trophozoites to the lysis mediated by serum complement (Braga et al. 1992; Espinosa-Cantellano and Martinez-Palomo 2000). However, the C1q inhibitory effect of CRT in E. histolytica/ E. dispar has not been documented until now, studies in that direction are ongoing. However, there is recent information that recombinant CRT of E. histolytica is not able to inhibit the C1q component of human complement (Teixeira and Huston 2008). Finally, the present work adds evidence about the presence of another important ER protein in E. histolytica HM1:IMSS trophozoites. The highly immunogenic character of this protein for the human host, particularly in invasive amoebiasis (González et al. 2002), makes it necessary to go deeper into the role of CRT in the complex pathogenic mechanisms of E. histolytica. For that purpose, the in vivo experimental models are the first necessary approach.
Acknowledgments Mr. Enrique González is a PhD fellow in Biological Sciences of Universidad Autónoma Metropolitana, Plantel Xochimilco in México City. We are very grateful to Dr. Victor Tsutsumi for his comments and suggestions on the original manuscript. We appreciate the collaboration of Mr. Jaime Escobar-Herrera for his help with the confocal assays, and. Mr. Mario Nequiz- Avendaño, for the Entamoeba histolytica axenic culture. We would also like to acknowledge Mr. Marco Gudiño for the informatics and art design and the secretarial assistance of Mrs. Ma. Elena Ortiz. Financial support The present work was partially supported by the following grants: IN206405-3, IN226806, and IN204208 from PAPIIT (DGAPA), National Autonomous University of Mexico (UNAM); Grant PE200105 from PAPIME (DGAPA) UNAM; and Grant SEP– CONACYT no. 79220 from the National Council for Science and Technology (CONACyT).
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