Experimental Parasitology 129 (2011) 65–71

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Experimental Parasitology

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Differentiation of Entamoeba histolytica: A possible role for enolase

Norma Cristina Segovia-Gamboa, Patricia Talamás-Rohana, Amelia Ángel-Martínez,Febe Elena Cázares-Raga, Arturo González-Robles, Verónica Ivonne Hernández-Ramírez,Adolfo Martínez-Palomo, Bibiana Chávez-Munguía ⇑Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Mexico City, Mexico

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 June 2010Received in revised form 29 April 2011Accepted 9 May 2011Available online 18 May 2011

Keywords:Entamoeba histolyticaEncystationEnolase

0014-4894/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.exppara.2011.05.001

⇑ Corresponding author. Address: Department ofPathogenesis, Center for Research and Advanced StudZacatenco, 07360 Mexico City, Mexico.

E-mail address: bchavez@cinvestav.mx (B. Chávez

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experi-mental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles sim-ilar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only inamebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody(B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histoly-tica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continu-ously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen wasidentified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blotand mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigenwas localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. TheB4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. Theseresults suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed inthe unfavorable liver environment that could be interpreted as an attempt to initiate the encystationprocess.

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1. Introduction

Intestinal and liver amebiasis produced by the protozoan para-site Entamoeba histolytica continues to be an important healthproblem in some developing countries. The availability of an ade-quate medium for the continuous cultivation of trophozoites underaxenical conditions (Diamond et al., 1978) has facilitated numer-ous studies of the motile form of the parasite. However, as conse-quence of continuous cultivation, a significant decrease of thevirulence of amebas has been reported in many laboratories. Theintrahepatic inoculation and later recuperation of trophozoitesfrom amebic hepatic lesions has been utilized as an effective meth-od for virulence reactivation (Das and Ghoshal, 1976). Recently,Bruchhaus et al. (2002) reported that E. histolytica trophozoitesrecovered from experimental amebic liver lesions showed differen-tial expression of several genes, encoding proteins specificallyassociated mostly with stress response, regulation of transcriptionand vesicular trafficking, which were upregulated in comparisonwith amebas grown continuously under axenic conditions. In addi-tion, we have found that the cytoplasm of liver-recovered tropho-

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Infectomics and Molecularies, Av. IPN 2508, San Pedro

-Munguía).

zoites contains vesicles with densely packed fibrilar material(Chávez-Munguía et al., 2004), morphologically similar to thoseobserved during the encystation process in other biphasic microor-ganisms (Gillin et al., 1996; Chávez-Munguía et al., 2003, 2005).

In contrast to a wealth of information concerning the biology ofthe motile form of the parasite, the trophozoite, the process of cystformation of E. histolytica remains basically unknown, in spite ofthe fact that the cysts are responsible for the transmission of theinfection. One of the obstacles is the lack of an experimental proce-dure that allows a massive production of E. histolytica cysts. In spiteof this limitation, some progress in this matter has been achieved.Chitin, an N-acetyl-D-Glucosamine polymer, was identified as theprincipal component of the cyst wall in both E. histolytica and inthe reptilian parasite Entamoeba invadens (Arroyo-Begovich et al.,1980; García-Zapién et al., 1999). The participation of two chitinsynthases in the construction of the human parasite cyst wall hasalso been reported (Van Dellen et al., 2006). Besides, two abundantencystation-specific proteins have been identified in E. histolytica;one of them is the remodeling enzyme chitinase (Ghosh et al.,1999; Van Dellen et al., 2002) and the other is a protein named Ja-cob, a cyst wall resident glycoprotein composed of five randomlyarranged chitin-binding domains (Frisardi et al., 2000; Van Dellenet al., 2002; MacFarlane et al., 2005). In addition, chitinase genehomologues and two lectins named Jessies were also localized inputative secretory vesicles of E. histolytica (Van Dellen et al.,

66 N.C. Segovia-Gamboa et al. / Experimental Parasitology 129 (2011) 65–71

2002). More recently Aguilar-Díaz et al. (2010) have producedin vitro E. histolytica cyst-like structures by treating trophozoiteswith hydrogen peroxide and trace amounts of several cations.

Using E. invadens as encystation model, ultrastructural studieshave shown that the cyst wall delivery to the cell surface takesplace through trafficking of specific secretory vesicles (Chávez-Munguía et al., 2003; De Souza, 2006). Through the use B4F2mAb (Segovia-Gamboa et al., 2010) we were able to determine thatin E. invadens both these vesicles and the cyst wall contain enolase.In the present study we have found that enolase is also present inthe cytoplasmic vesicles produced in E. histolytica trophozoitesrecovered from amebic liver lesions.

Fig. 1. (A) ELISA assays with the B4F2 mAb. EhR72 = recognition of E. histolyticatrophozoite extracts recovered from amebic liver lesions 72 h post-infection.NORMAL = E. histolytica trophozoite extracts in continuous culture. Cysts = extractsof mature E. invadens cysts. Experiments were done three times in triplicate. (B)Western blot assay with B4F2 culture supernatant identifies a protein of 48 kDa asthe antigen in E. histolytica trophozoites recovered from liver lesions. Lane1 = molecular weight markers; lane 2 = soluble fraction of trophozoites E. histolyticaliver-recovered at 72 h; lane 3 = trophozoites of E. histolytica in continuous culture;lane 4 = soluble fraction of mature cysts of E. invadens. Western blot of equivalentsamples (upper panel).

2. Materials and methods

2.1. Cultivation of E. histolytica and isolation of trophozoites fromhepatic lesions

E. histolytica trophozoites (HM-1-IMSS strain) were axenicallygrown in borosilicate tubes at 36 �C in complete TYI-S-33 medium(Diamond et al., 1978) supplemented with 10% bovine serum andharvested during the logarithmic phase of growth (48 h). Malehamsters (Meriones ungiculatus) from CINVESTAV-IPN animal facil-ities, complying with international regulations, were used to pro-duce liver abscesses. Trophozoites (1 � 106) in the logarithmicphase of growth in 200 ll of culture medium were inoculated inthe left lobe of hamster livers. Fragments of liver lesions wererecovered at different times post infection, under sterile conditions(12, 24, 48, 72 and 96 h), and incubated in TYI-S-33 complete cul-ture medium as above, supplemented with 1000 U penicillin and100 lg streptomycin. When recovered parasites reached the firstlogarithmic phase of growth, they were harvested and processedaccording to the required analysis.

2.2. Fluorescence microscopy

Kinetic analysis of liver-recovered E. histolytica trophozoiteswere performed by collecting samples at 12, 24, 48, 72 and 96 hpost-infection, fixed with 4% paraformaldehyde for 15 min at roomtemperature and permeabilized with 0.05% Triton X-100 in PBS. E.histolytica mature cysts obtained from patients were also fixedwith 4% paraformaldehyde for 30 min at room temperature. Allsamples were then blocked with 10% BSA/PBS for 1 h. Later theywere incubated with the B4F2 mAb (1:50 dilution). A goat anti-mouse IgG conjugated to FITC was used as secondary antibody(1:150 dilution). Incubation lasted for 1 h at 37 �C. Samples weremounted in Vecta-Shield medium (Vector Laboratories Inc., Burlin-game, CA, USA) and observed under an Axiophot photo-microscope(Carl Zeiss Germany) equipped with a Zeiss AxioCam MRc digitalcamera (Carl Zeiss Vision GmbH, Germany).

2.3. Electrophoresis and Western blot

For antigen preparation, recovered trophozoites (4 � 106) wereresuspended in 400 ll of buffer (Tris–HCl 0.05 M, NaCl 0.15 M withprotease inhibitors), after vortex they were centrifuged, and thesupernatant separated. Extracts were resuspended in sample buf-fer with 2-b mercaptoethanol, and boiled for 3 min. Protein ali-quots of each sample (50 lg/10 ll) were loaded onto 10% sodiumdodecyl sulfate-polyacrylamide gels. For protein analysis, gelswere stained with silver. For Western blotting, cellular proteinswere transferred to nitrocellulose (NC) membranes (Bio-Rad 162-0115) (Towbin et al., 1979). Antigen-antibody reactions were de-tected using enhanced chemiluminescence (ECL-Bio-Rad). Culturesupernatant (1:500), purified B4F2 mAb (1:5000) or a commercial

anti-actin antibody (1:5000) was used as primary antibodies. Cystsextracts were processed as reported previously (Segovia-Gamboaet al., 2010).

2.4. Two-dimensional gel electrophoresis and mass spectrometry

Analysis of proteins by two-dimensional (2D) polyacrylamidegel electrophoresis was done using immobilized pH gradient(IPG) strips for the first dimension (Görg et al., 2000). E. histolyticarecovered trophozoites (1 � 106) were resuspended in 200 ll of ly-sis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, 2% Phar-malyte pH 4–7), with proteases inhibitors; after sonication (20pulses � 2 min each) they were centrifuged, and the supernatantseparated. The trophozoite extract was precipitated with 2DClean-Up kit (Amersham Biosciences, GE Healthcare). The proteinpellet was resuspended in 250 ll of rehydration solution (7 M urea,2 M thiourea, 2% CHAPS, 2% pharmalyte pH 4–7, and 1% bromophe-nol blue) with protease inhibitors. Trophozoites extracts were ap-

Fig. 2. Identification of the B4F2-specific antigen by two-dimensional electropho-resis and Western blott. E. histolytica trophozoites liver-recovered at 72 h post-infection. Soluble proteins were separated in strips at pH 4–7 and 10% SDS–PAGE.The gel was stained with Bio-Safe Coomassie (upper panel). A parallel gel wasblotted onto NC membrane and incubated with B4F2 mAb (lower and right panel).A protein spot recognized by the B4F2 mAb was excised and identified by ESI-LCMS/MS.

Fig. 3. (A) Subcellular distribution of the enolase by indirect immunofluorescence asstrophozoites of liver-recovered E. histolytica. Bars = 10 lm. (B) Subcellular distribution of12 h (1), 24 h (2), 48 h (3), 72 h (4) 96 h (5), trophozoites in continuous culture (6). Bar

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plied to 13 cm Immobiline DryStrips gels pH 4–7 (GE HealthcareBiosciences) by rehydration, and run for isoelectric focusing (IEF)in an Ettan IPGphor 3 (GE Healthcare). After IEF, DryStrips gelswere stored at �75 �C until required. Gel equilibration was per-formed immediately prior to the second dimension in 10% SDS–PAGE, and was stained with Bio-Safe Coomassie (Bio-Rad). A rep-lica gel was electrotransferred onto a NC membrane, reacted withB4F2 mAb, and developed using ECL (Rosewell and White, 1978).The spots that reacted with B4F2 mAb were excised from a Coo-massie stained 2D gel digested with trypsin and sent to ColumbiaUniversity to be analyzed by mass spectrometry (ESI-LC/MS/MS).Peptides identified were matched at NCBI using Mascot program(http://www.matrixscience.com/search form select.html).

2.5. Immunoelectron microscopy

Liver-recovered E. histolytica trophozoites were harvested andwashed three times with PBS and once with serum-free DMEM.Cells were fixed in 4% paraformaldehyde and 0.1% glutaraldehydein serum-free DMEM for 1 h at room temperature. Samples wereembedded in LR White and polymerized under UV at 4 �C over-night. Sections were obtained and mounted on formvar-coverednickel grids. Later they were incubated with the B4F2 mAb (1:10dilution). A goat anti mouse IgG conjugated to 20 nm gold particles

ays using the B4F2 mAb. (1) E. histolytica trophozoites in continuous culture; (2)the enolase in trophozoites of liver-recovered E. histolytica in samples recovered at

s = 10 lm.

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was used as secondary antibody (1:40 dilution). Immunolabelingwas carried out at room temperature. Thin sections were observedwith a Morgani 268 D Philips transmission electron microscope(FEI Company, Eindhoven, The Netherlands).

2.6. Semi-quantitative RT-PCR analysis

Total RNA was isolated using Trizol reagent (Invitrogen) fromtrophozoites in continuous culture, and from trophozoites recov-ered from liver lesions. The total RNA was treated with RNasefree-DNaseI (Ambion) according to the manufacture’s protocol.The RNA was precipitated with isopropanol, rinsed with 70% etha-nol, and redissolved in 20 ll of DEPC-treated water. RNA concen-tration was determined by the DNA/RNA ratio through readingstaken at 260/280 nm. Total RNA (5 lg) from each sample was tran-scribed to cDNA. For PCRs 2 ll aliquots of cDNA were amplifiedusing enolase specific primers (forward 50-TCA TGT GTT CCA TCAGGA GC-30 position 260–279; and reverse 50-CTG CAG CTC TGAAAA TTG AC-30 position 505–524) to obtain a fragment of 265 bp.Amplification was performed in a MyCycler (Bio-Rad) using the fol-lowing parameters: 94 �C for 2 min to denature, 35 amplificationcycles (95 �C, 1 min; 60 �C, 1 min; 72 �C, 2 min); and an end exten-sion at 72 �C for 8 min. RT-PCR products were analyzed on a 2%agarose gel with ethidium bromide and quantified using QuantityOne Software (Bio-Rad). GAPDH was used as a control.

3. Results

The present results show that the B4F2 mAb, produced againstthe cyst wall of E. invadens, recognizes by ELISA an antigen presentin soluble extracts of liver-recovered E. histolytica trophozoites(Fig. 1A).

In order to analyze whether the recognized antigen by ELISAcorresponds to the same molecule identified by the mAb in ency-sting E. invadens, a Western blot assay was carried out. Resultsshowed that the B4F2 mAb recognized specifically a 48 kDa proteinin soluble extracts of E. histolytica trophozoites recovered from theliver abscesses at 72 h post infection (Fig. 1B, lane 2). A very lightrecognition by the mAb was found in trophozoites continuouslycultured under axenical conditions (Fig. 1B, lane 3). The antigen

Fig. 4. (A) a, Silver staining of processed samples. C, E. invadens cysts extract; 0, long termtrophozoites of E histolytica; b, Western blot assay using purified B4F2 (1:5000 dilution)Molecular weight markers are shown on the right of a and c. d, Densitometric analyindependents experiments. (B) Expression profile of enolase mRNA in E. histolytica trophamebas were separated on a 2% gel agarose stained with ethidium bromide a1, A band ofcontrol for the samples a2. b, Densitometric analysis of the amounts of enolase RT-PCR

recognized by the mAb was similar in molecular weight to thatof E. invadens cysts (Fig. 1B, lane 4).

To determine if the protein recognized by the B4F2 mAb in li-ver-recovered E. histolytica trophozoites corresponds to the proteinrecognized in E. invadens, previously identified by us as enolase, aWestern blot assay was performed (Fig. 2, lower right panel) wereperformed after two-dimensional electrophoresis of soluble ex-tracts of trophozoites (Fig. 2, lower left panel). A strong signal,likely corresponding to a protein with an apparent molecularweight of 48 kDa and a pI of 5.87 was detected. The putative pro-tein was processed by ESI-LC-MS/MS mass spectrometric analysis.Protein identification was achieved in a database search using theMascot search program. The sequence of the spot matched with E.histolytica and Entamoeba dispar enolase, a protein with a Mr of48 kDa and a pI of 5.87, showing a 25% of sequence coverage,and a score of 344.

The subcellular distribution of the enolase in recovered tropho-zoites was analyzed by immunofluorescence assays carried outwith the B4F2 mAb. Results showed small fluorescent vesicle-likestructures dispersed throughout the cytoplasm of liver-recoveredtrophozoites, after incubation with the monoclonal antibody(Fig. 3A, 2). Similar vesicles were absent in trophozoites obtainedfrom continuous cultures (Fig. 3A, 1). To determine if the enolaseis expressed early after infection, a kinetic analysis was performedwith liver-recovered trophozoites from 12 until 96 h post infection.Immunofluorescence assays showed fluorescent vesicle-like struc-tures in the cytoplasm of liver-recovered trophozoites, after incu-bation with the monoclonal antibody from 12 h until 96 h postinfection (Fig. 3B, 12 h (1), 24 h (2), 48 h (3), 72 h (4), 96 h (5),and trophozoites in continuous culture (6).

By Western blot the B4F2 mAb recognized enolase as early as12 h until 96 h post infection (Fig. 4Ab). The antigen was also de-tected in trophozoites in continuous culture for long periods(Fig. 4Ab, T0), but the recognition was approximately 50% of theamount present in recovered trophozoites (Fig. 4Ad). E. invadenscysts extracts were used as positive control (Fig. 4Ab, c). An anti-actin antibody was used as loading control using the same NCmembrane. Fig. 4Aa shows the silver staining pattern of processedsamples.

To determine if enolase mRNA expression was induced as infec-tion progressed, the level of enolase mRNA was determined

cultured E. histolytica trophozoites extract; 12, 24, 48, 72 and 96 h, liver-recovered, and c, Western blot assay using anti-actin (1:5000 dilution) as primary antibodies.sis of western blot assays shown in b and c, results are representative of three

ozoites recovered at different times. RT-PCR products obtained from liver recovered265 bp corresponding to an enolase fragment is shown. GAPDH was used as loadingfragments.

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(Fig. 4Ba). A fragment of the expected size (265 bp) was detected incontinuous cultured trophozoites and also in liver-recovered tro-phozoites. By densitometric analysis, we found that cultured tro-phozoites had a basal level of enolase mRNA and expression levelof the mRNA was induced in liver-recovered trophozoites since12 h post infection and remained without further significantchanges (Fig. 4Bb).

The ultrastructural localization of the enolase in recovered E.histolytica trophozoites was established by transmission immuno-electron microscopy (Fig. 5A). As expected, enolase was specificallylocated inside vesicles containing densely packed fibrilar material(Fig. 5A, insert and B). When only the secondary antibody was usedas control, the labeling was negative (Fig. 5C).

Moreover, the B4F2 mAb recognized enolase at the cyst wall ofE. histolytica mature cysts obtained from human stools as analyzedby immunofluorescence (Fig. 6A) and transmission immunoelec-tron microscopy (Fig. 6B).

Fig. 5. Immunoelectron microscopy. Localization of the antigen recognized by B4F2 mAblabeled cytoplasmic vacuole with fibrilar content. Insert. Higher magnification of the vControl without primary antibody (B4F2) a vesicle. Bars = 500 nm.

4. Discussion

The production of cysts from axenically cultured E. histolytica tro-phozoites has eluded efforts of various groups interested in the biol-ogy of the encystation process. Previous attempts have included avariety of stress conditions, such as carbon source deprivation, os-motic shock, or a combination of the two, that induce encystationin E. invadens (Avron et al., 1986; Bailey and Rengpien, 1980; Váz-quez de Lara-Cisneros and Arroyo-Begovich, 1984). Others havetried unsuccessfully the addition of cofactors (Mg2+ Mn2+ andCo2+) (Campos-Góngora et al., 1997) or bacteria, such as Escherichiacoli and Enterococcus fecalis (Said-Fernández et al., 1993; Morales-Vallarta et al., 1997; Barrón-González et al., 2008). More recentlythe in vitro induction of E. histolytica cyst-like structures was re-ported in axenicaly cultured trophozoites treated with hydrogenperoxide and trace amounts of various cations (Aguilar-Díaz et al.,2010). Several ultrastructural features as well as the surface staining

in liver-recovered E histolytica trophozoites at 72 h post- infection. (A) A positively-acuole. (B) Another example of a B4F2 positively-labeled cytoplasmic vacuole. (C)

Fig. 6. Subcellular distribution and localization of the B4F2 positive antigen in mature cysts of E. histolytica obtained from a human carrier sample. A1 = immunofluorescenceindirect assay. A2 = phase contrast image of the same cyst. Bars = 10 lm. (B) Immunoelectron microscopy assay. The B4F2 mAb recognized contents at the cyst wall of an E.histolytica cyst obtained from human carrier. Bar = 500 nm.

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with calcofluor of these cyst-like structures tend to support theircyst nature. However, further definition of the best incubation con-ditions, as well as a more detailed study of the fine structure of thewall components of these cyst-like cells is required.

Our group described the appearance of cytoplasmic vesicles inE. histolytica trophozoites recovered from amebic liver lesions(Chávez-Munguía et al., 2004), that showed fluorescent reactionwith calcofluor white m2r and resemble ultrastructurally the ency-station vesicles found in Giardia lamblia (Gillin et al., 1996), inE. invadens and in Acanthamoeba castellani (Chávez-Munguíaet al., 2003, 2005).

In view of the ultrastructural similarity between the cyst wallsof E. invadens and E. histolytica, we have used in the present studythe B4F2 mAb that specifically recognizes the E. invadens cyst wall,as a tool to further characterize the vesicles with fibrilar contentpresent in E. histolytica trophozoites recovered from amebic liverlesions. The present results demonstrate that in these amebas themAb recognized a protein of 48 kDa, which was further identifiedas enolase. Both protein and mRNA increased at 12 h post-infection, suggesting that enolase synthesis is regulated at thetranscriptional and translational levels.

By immunofluorescence microscopy the B4F2 mAb localizedenolase in cytoplasmic vesicles in E. histolytica liver-recovered tro-phozoites. These vesicular structures are not present either inE. histolytica trophozoites cultured in axenic conditions for longperiods, or in trophozoites submitted to other stress conditions(Akbar et al., 2004; Debnath et al., 2005; Ehrenkaufer et al.,2007; McGugan and Temesvari, 2003). The specific recognition ofenolase by the B4F2 mAb in cytoplasmic vesicles was confirmedby transmission electron microcopy. Furthermore, this antibodyalso recognized an antigen, most probably enolase in the wall ofmature cysts isolated from human carriers.

Our group first reported a non glycolytic function of enolase inE. invadens (Segovia-Gamboa et al., 2010). The results here pre-sented extend this finding to trophozoites of E. histolytica recentlyobtained from liver amebic lesions.

Enolase has been reported in E. histolytica as a glycolytic en-zyme and the respective gene was cloned (Hidalgo et al., 1997).

A crucial role of enolase in the modulation of cytosine-5 methyl-transferase 2 activity in the nucleus of this human parasite was re-cently reported (Tovy et al., 2010). In addition, its immunogenicnature has been established in a patient with amebic liver abscessthrough the presence of SlgA against this protein in the patient sera(Carrero et al., 2000). According to the sequence of the E. histolyticaenolase (EhEnl-1) gene (Ehehl-1), the predicted protein has 436amino acids (47.3 kDa) and is about 60% identical to enolase ofrat, Drosophila and Saccharomyces cerevisiae.

In Candida albicans, enolase is the main protein associated withglucans, where it is used as a scaffold for the formation of the cellwall (Chaffin et al., 1998). Enolase lacks a secretory signal for clas-sical secretion, however, it has been clearly demonstrated that theprotein is able to reach the cell wall in C. albicans, suggesting thatsome moonlighting proteins might bind through either glucans ornon specific protein interaction (Chaffin, 2008). Another alterna-tive transport mechanism for proteins lacking the signal sequencecould be to use of post-Golgi vesicles, carrying classical pathwayproteins towards the plasma membrane (De Groot et al., 2005).Endosome recycling may be another possibility of transport(Nickel, 2005). In Saccharomyces cerevisiae Decker and Wickner(2006) have suggested that enolase may be related with the trafficof proteins to vacuoles and with fusion of these vacuoles to theplasma membrane. In A. castellanii enolase was expressed, amongother cyst specific proteins (Bouyer et al., 2009), suggesting thatenolase may be related to cellulose biosynthesis.

Given that in our Entamoeba study enolase was localized both inthe cytoplasmic vesicles and in the cyst wall, this would suggestthat this protein uses these vesicles for transport and incorporationto the cell wall. However, more studies are necessary in define andbetter understand this transport mechanism.

Considering that the encystation process occurs only in theintestinal environment we propose the hypothesis that in the liver,parasites may be exposed to stress conditions which activate genesrelated with virulence and probably also genes related with theencystment process.

Our results suggest that the fibrilar content vesicles presentin the cytoplasm of E. histolytica trophozoites recovered from

N.C. Segovia-Gamboa et al. / Experimental Parasitology 129 (2011) 65–71 71

hamster liver lesions may correspond to encystation-specific ves-icles described in other biphasic microorganisms. Results alsosuggest that E. invadens and E. histolytica share at least oneimmunodominant epitope in the enolase which belongs to a cystwall component.

Acknowledgments

We thank to Ana Ruth Cristóbal Ramos and Lizbeth Salazar Vil-latoro for their technical assistance. This study was financially sup-ported by Grant No. PIFUTP08-157 to Dr. P. Talamás-Rohana andDr. B. Chávez-Munguía.

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