ORIGINAL PAPER

The small GTPase EhRabB of Entamoeba histolyticais differentially expressed during phagocytosis

Mario Hernandes-Alejandro & Mercedes Calixto-Gálvez &

Israel López-Reyes & Andrés Salas-Casas &Javier Cázares-Ápatiga & Esther Orozco &

Mario A. Rodríguez

Received: 19 July 2012 /Accepted: 23 January 2013 /Published online: 12 February 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract It has been described that the pathogenicity ofEntamoeba histolytica is influenced by environmental con-ditions and that transcription profile changes occur duringinvasion, suggesting that gene expression may be involvedin the virulence of this parasite. However, the molecularmechanisms that are implicated in the control of gene ex-pression in this microorganism are poorly understood. Here,we showed that the expression of the EhRabB protein, asmall GTPase involved in phagocytosis, is modified throughthe interaction with red blood cells. By ELISA, Westernblot, and immunofluorescence assays, we observed that theexpression of EhRabB diminished after 5 min of the inter-action of trophozoites with red blood cells, but protein levelwas recovered at subsequent times. In the EhRabB aminoacid sequence, we found two lysine residues that could betarget for ubiquitin modification and trigger the degradationof this GTPase at early times of phagocytosis. The analysisof the expression of the EhrabB mRNA showed that theinteraction of trophozoites with red blood cells produces a

drastic diminishing in its half-life. In addition, promoterassays using the chloramphenicol acetyltransferase reportergene and electrophoretic mobility shift assays experimentsshowed that the URE1 motif located in the promoter regionof EhrabB is involved in the expression regulation of thisgene during phagocytosis. Moreover, the immunolocaliza-tion of the URE1-binding protein during phagocytosis indi-cated that the transcription of the EhrabB gene isdetermined, at least in part, by the translocation of thistranscription factor to nuclei. These results suggested thatthe expression of particular genes of this parasite is con-trolled at several stages.

Introduction

For all cells, the regulation of gene expression is a fundamen-tal mechanism to drive development, homeostasis, and adap-tation to the environment. In eukaryotes, any step of geneexpression may be controlled, from the transcription of DNAinto RNA to the post-translational modifications of the pro-teins. Transcription initiation is mediated by the cis-regulatoryelements located in gene promoters, the concerted action oftranscription factors along with the RNA polymerase II tran-scriptional machinery, and a diversity of co-regulators thatbridge the DNA-binding factors to the transcriptional machin-ery (Hirose and Manley 2000). Then, post-transcriptionalevents such as mRNA processing and nuclear export,mRNA stability, and microRNA-dependent modulation createa complex intracellular network contributing to determine theglobal levels of specific mRNAs (Moore 2005). Most mRNAregulatory elements are located within the 5′ and 3′ untrans-lated regions (UTRs), where they function as platforms for thebinding of numerous proteins and non-coding RNAs. The 5′-UTR is principally involved in controlling mRNA translation

M. Hernandes-Alejandro :M. Calixto-Gálvez :A. Salas-Casas :J. Cázares-Ápatiga : E. Orozco :M. A. Rodríguez (*)Departamento de Infectómica y Patogénesis Molecular, Centro deInvestigación y de Estudios Avanzados del IPN, A.P. 14-740,07000, Mexico, D.F., Mexicoe-mail: marodri@cinvestav.mx

I. López-ReyesCentro de Diagnóstico y Vigilancia Epidemiológica del DistritoFederal, Instituto de Ciencia y Tecnología del Distrito Federal,06010, Mexico, D.F., Mexico

Present Address:A. Salas-CasasInstituto de Ciencias de la Salud, Universidad Autónomadel Estado de Hidalgo, Carretera Actopan-Tilcuautla,Exhacienda la Concepción S/N,San Agustin Tlaxiaca, Hidalgo, Mexico

Parasitol Res (2013) 112:1631–1640DOI 10.1007/s00436-013-3318-2

(Pickering and Willis 2005), while the 3′-UTR regulates themultiple steps of mRNA metabolism and stability (Andreassiand Riccio 2009; Moore 2005). In addition, the covalentmodification of proteins by ubiquitin and ubiquitin-like pro-teins is involved in the regulation of numerous cellular path-ways cells (Kravtsova-Ivantsiv and Ciechanover 2011). Inmany cases, the modification is followed by the targeting ofthe tagged proteins to proteasomal or lysosomal degradation,thus terminating their function (Kravtsova-Ivantsiv andCiechanover 2011).

In particular, parasites should perform a strict control onthe expression of genes involved in their pathogenicity,differentiation, or immune evasion, and the expression ofthese genes could be modulated by the target cell–parasiteinteraction or by some compounds present in the hosttissues. For instance, bile induces the excystation ofEntamoeba invadens cysts (Mitra et al. 2010) and alsostimulates the expressions of genes that produce the energyrequired by newly excysted juvenile Clonorchis sinensis tomigrate to the bile duct and to modulate the regulatorysignals of cell proliferation associated with adult develop-ment (Kim et al. 2008). On the other hand, the release ofreactive oxygen and nitrogen species, as part of the hosts’innate immune response against infections, induces the ex-pression of several parasite genes that are needed to surviveinto their hosts and contributes to disease outcome (Kim etal. 2012; Rastew et al. 2012).

The protozoan Entamoeba histolytica is the etiologicagent of human amebiasis, a disease that affects up to 50million people worldwide each year and causes from 40,000to 100,000 deaths annually (WHO 1997). The molecularmechanisms participating in the parasite invasiveness arenot completely understood, but it has been reported thatthe pathogenicity is influenced by environmental conditionssuch as bacteria flora and cholesterol (Bracha and Mirelman1984; Meerovitch and Ghadirian 1978). Moreover, the tran-scription changes of several genes occur during invasion(Bruchhaus et al. 2002; Gilchrist et al. 2006; Gonzalez etal. 2011). Thus, modification in gene expression may beinvolved in the virulence of E. histolytica. However, despitesome studies that have been performed to characterize thegene expression regulation of a few genes (Gomez et al.2010), the molecular mechanisms that are implicated in thecontrol of the gene expression in this parasite are poorlyunderstood.

EhRabB is a Rab GTPase of E. histolytica located closeto plasma membrane and in phagocytic mouths when troph-ozoites are incubated with red blood cells (RBCs)(Rodriguez et al. 2000), suggesting that this protein is in-volved in phagocytosis, an event related to the virulence ofthe parasite (Orozco et al. 1983). Functional assays showedthat the transcription of EhrabB is coordinately regulated bydifferent cis-elements that are situated in its gene promoter.

In basal conditions, the transcription of the EhrabB gene isregulated positively and negatively by two cis-elements(Romero-Diaz et al. 2007). An URE1 sequence situatedbetween positions −428 and −413 with respect to the firstinitiation transcription site activates the gene transcription(Romero-Diaz et al. 2007), and recently, we identified apolypeptide containing staphyloccocal nuclease and Tudordomains as the URE1-binding protein (EhURE1BP)(Calixto-Galvez et al. 2011). On the other hand, the elementthat negatively controls the transcription of the EhrabB genewas situated in a fragment of 67 bp, between positions −491and −428 (Romero-Diaz et al. 2007).

In addition, the expression of EhrabB is modified bysome environmental stimuli. Functional assays with a con-struct that includes seven putative heat shock elements ofthe EhrabB gene promoter showed an increase of twice thechloramphenicol acetyltransferase (CAT) activity of heatshocked trophozoites with respect to parasites maintainedat 37 °C (Romero-Diaz et al. 2007). In concordance, theEhrabB transcript showed a significant augment in tropho-zoites submitted to heat shock (Romero-Diaz et al. 2007).Additionally, immunofluorescence assays showed an appar-ent variation in the amount of the EhRabB protein at differ-ent times of phagocytosis (Rodriguez et al. 2000).

In this study, we showed that the expression of EhRabB,at protein and mRNA levels, is downregulated at the earlytimes of the phagocytosis of RBCs. The decrease in thelevels of the EhrabB transcript during phagocytosis seemsto be a consequence of a decrease in the mRNA half-life. Inaddition, we hypothesized that there are also a diminution inthe transcription of EhrabB due to a reduction in the amountof EhURE1BP located in nuclei.

Material and methods

E. histolytica cultures

The trophozoites of E. histolytica clone A (strain HM1:IMSS;Orozco et al. 1983) were axenically cultured in TYI-S-33medium and harvested as described (Diamond et al. 1978).

ELISA, Western blot, and immunofluorescence

For all experiments, trophozoites were incubated with RBCs(1:100) at 37 °C during 5, 10, and 30 min. After each time,the non-ingested RBCs were lysed by 10-min incubationwith distilled water. Then, the total extracts of trophozoiteswere obtained in the presence of protease inhibitors(Complete® Mini; Roche, Mannheim). At time 0, we usedtrophozoites that were not incubated with RBCs.

For the enzyme-linked immunosorbent assays (ELISA),96-well plates (Costar) coated with 35 μg of proteins

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obtained from trophozoites at different times of phagocyto-sis were incubated for 2 h at room temperature with specificantibodies against EhRabB (1:2,000) (Rodriguez et al.2000). Then, the plates were incubated for 1 h at 37 °C witha mouse anti-rabbit IgG secondary antibody conjugated tohorseradish peroxidase (1:10,000; Invitrogen). The reactionwas developed as described (Engvall and Perlmann 1971)and measured in an ELISA reader (ICN, MS2) at 490 nm.An anti-actin antibody was used as an internal control.These experiments were performed three times by duplicate.

For Western blot assays, total extracts from trophozoiteswere separated by 12 % SDS-PAGE and transferred tonitrocellulose filters. Then, membranes were incubated withantibodies against EhRabB (1:2,000), followed by incuba-tion with a mouse anti-rabbit IgG secondary antibody con-jugated to horseradish peroxidase (1:10,000; Invitrogen).Finally, the antibody detection was developed by chemilu-minescence (ECL, GE Healthcare). As internal control, thesame membranes were probed with antibodies against actin.Relative intensities were documented and analyzed bydensitometry.

For immunofluorescence, trophozoites grown on cover-slides were incubated at 37 °C during different times withhuman RBCs (1:100). Then, samples were fixed with 4 %paraformaldehyde and permeabilized with 0.5 % Triton X-100 for 30 min at room temperature. Samples were thenincubated with 1 % BSA for 1 h at room temperature andincubated overnight at 4 °C with antibodies against EhRabB(1:500) or against EhURE1BP (1:6,000) (Calixto-Galvez etal. 2011). Then, cells were incubated for 1 h at 37 °C with afluorescein-labeled secondary antibody (1:1,000; Zymed).Nuclei were counterstained with 30 nm of 4′,6-diamino-2-phenilindole (DAPI) for 5 min. Samples were mounted withmedium for fluorescence (VECTASHIELD; VectorLaboratories) and examined through a confocal microscope(Leica TCS SP2). Observations were performed in14 planesfrom the top to the bottom of each sample, and the distancebetween scanning planes was 1 μm.

Real-time RT-PCR

To evaluate the amount of the mRNA of EhrabB duringphagocytosis, total RNA was obtained from trophozoites atdifferent times of phagocytosis using the TRIzol Reagent(Gibco BRL) according to the manufacturer’s recommenda-tions. Then, cDNA was synthesized using an oligo(dT)primer (Invitrogen), and PCR was performed utilizing spe-cific primers for EhrabB and 18S rRNA (used as normaliz-er) (Romero-Diaz et al. 2007). Quantitative amplifications(qRT-PCR) were performed in the 7300/7500/7500 FastReal-Time PCR System (Applied Biosystems) using theSYBR Green PCR Master Mix kit (Applied Biosystems)under the conditions previously described (Romero-Diaz et

al. 2007). The two replicates of each assay were analyzed intriplicate. Relative quantification was performed using thedelta–delta Ct method (Livak and Schmittgen 2001).

EhrabB mRNA stability assays

The half-life of the EhrabB transcript was determined asdescribed (Lopez-Camarillo et al. 2003; Seguin et al. 1995).Briefly, actinomycin D (Roche Molecular Biochemicals)was added to the cultures to reach a final concentration of10 μg/ml, and cells were incubated for 1 h at 37 °C. Then,trophozoites were incubated with RBCs (1:100) at 37 °C.Next, total RNA was isolated at different times, and cDNAsynthesis and reverse transcription (RT)-PCR were per-formed as described above. EhrabB mRNA levels werenormalized with respect to the 18 rRNA amount. In theseestimations, the EhrabB mRNA quantity from trophozoitesin the absence of RBCs was taken as 100 %.

Transient transfection and CAT assays

To analyze the promoter activity during phagocytosis, weused the pRab428 construction, which displayed the greatestCAT activity on functional assays and contains the transcrip-tional activator URE1 (Romero-Diaz et al. 2007). As posi-tive control, we used the pA5′A3′CAT vector that containsthe E. histolytica actin gene promoter (Nickel and Tannich1994) and as negative control the reporter vector withoutpromoter region (pBSCAT-ACT).

The transfection of trophozoites was carried out by elec-troporation as described (Nickel and Tannich 1994). CATactivity was determined by two-phase diffusion assays asdescribed (Gomez et al. 1998), incubating 100 μg ofextracts from trophozoites and 1.25 mM chloramphenicolwith [14C]-acetyl CoA (PerkinElmer) within 4 h. The back-ground activity displayed by trophozoites transfected withthe promoter-less vector was subtracted from activityobtained from cells transfected with each construct.Activities were expressed as relative activity with respectto that obtained from trophozoites transfected with the plas-mid pA5′A3′CAT. The enzymatic activity for each constructwas assayed at least three times by duplicate.

Electrophoretic mobility shift assays

A double-stranded oligonucleotide corresponding to posi-tion −428 to −412 (5′-TCTTAAATATTTGGAAC-3′) ofEhrabB and containing the URE1 motif (Romero-Diaz etal. 2007) was labeled at its 5′ end with T4 polynucleotidekinase (Invitrogen) and [γ-32P]ATP following standard pro-cedures (Sambrook and Russell 2001). Then, probe (1 ng)was incubated at 4 °C for 10 min with nuclear extracts(50 μg) in binding buffer (60 mM KCl, 1 mM DTT,

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1 mM EDTA, 1 mM MgCl2, 4 mM Tris–HCl, 1.2 mMHEPES, pH7.9, 10 % glycerol) in the presence of 1 mg ofpoly[d(I-C)] (Gomez et al. 1998). Competition assays wereperformed using as specific competitor a 150-fold excess ofthe same unlabeled fragment or as unspecific competitor a350-fold excess of an unrelated double-stranded oligonucle-otide. DNA–protein complexes were separated on 6 % non-denaturing polyacrylamide gels and visualized with a phos-phoimager analyzer (Bio-Rad).

Statistics

Data were expressed as the mean of independent experi-ments ± SD for each group. Statistical significance wasdetermined by Student’s t test, and a difference of p<0.05was considered significant.

Results and discussion

Expression of the EhRabB protein during phagocytosis

The way by which host cells are killed and phagocytosed byE. histolytica follows a sequential model of adherence, cellkilling, an initiation of phagocytosis, and engulfment, andseveral molecules are involved in these events (Sateriale andHuston 2011). Vesicular trafficking plays an essential role inthe expression of virulence of this parasite, and this processis orchestrated by small GTPases, Rab proteins, which act asmolecular switches regulating the fusion of vesicles withtarget membranes through the conformational change

between active (GTP-bound) and inactive (GDP-bound)forms (Stenmark and Olkkonen 2001). Thus, Rab proteinshave an important role in the virulence of E. histolytica(Nozaki and Nakada-Tsukui 2006). EhRabB is a RabGTPase of E. histolytica that is translocated to plasmamembrane and phagocytic mouths during phagocytosis,and interestingly, the amount of EhRabB seems to be vari-able during the engulfment of human erythrocytes(Rodriguez et al. 2000). To confirm this finding, we ana-lyzed the protein level of EhRabB during the phagocytosisof RBCs using specific antibodies against this protein(Rodriguez et al. 2000). The data obtained from tropho-zoites in the absence of RBCs (time 0) were arbitrarily takenas 100 % of expression. In all experiments, antibodiesagainst actin were used as internal control.

By ELISA, we observed only about 20 % of the EhRabBexpression at 5 min of phagocytosis (Fig. 1a). After thistime, the quantity of EhRabB was sequentially recovered,and after 30 min of phagocytosis, we detected approximate-ly 80 % of expression with respect to that observed in theabsence of RBCs (Fig. 1a). In contrast, the expression ofactin was similar during the different times of phagocytosis(Fig. 1a), suggesting that the EhRabB expression variesduring phagocytosis.

These observations were corroborated by Western blotassays. In these experiments, the intensity of the 21-kDaband recognized by the antibodies against EhRabB de-creased after 5 min of interaction with RBCs, but the signalof this band enhanced at subsequent times of phagocytosisreaching almost the same level as that of the control troph-ozoites after 30 min of phagocytosis (Fig. 1b). On the

Fig. 1 The expression of EhRabB protein during the phagocytosis ofRBCs. The trophozoites of E. histolytica were incubated at 37 °C withRBCs at different times. Then, the expression of EhRabB was analyzedusing specific antibodies against it. As internal control, we used anti-bodies against actin to analyze the expression of this protein under thesame conditions. a ELISA. bWestern blot. c The relative expression of

EhRabB. The relative intensities of the bands detected by Western blotwere documented and analyzed by densitometry. The protein level ofEhRabB was normalized to the actin protein level of each sample, andthe value obtained in the absence of RBCs (time 0) was taken arbi-trarily as 1. Bars show the mean levels (±SD). Asterisks indicatesignificant difference (p<0.05) compared with the time 0

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contrary, the 45-kDa band recognized by antibodies againstactin showed approximately the same intensity at differenttimes of phagocytosis (Fig. 1b). Densitometric analysisshowed that the EhRabB protein expression was about 20,

60, and 80 %, with respect to the control, at 5, 10, and30 min of interaction with RBCs, respectively (Fig. 1c).

We also analyzed the expression of the EhRabB proteinduring phagocytosis by immunofluorescence assays. As

Fig. 2 The immunofluorescence microscopy of EhRabB duringphagocytosis. Trophozoites were grown in coverslides and incubatedat 37 °C with RBCs at different times. Then, samples were fixed,permeabilized, and reacted with anti-EhRabB antibody. Bound

antibody was detected with a FITC-conjugated secondary antibodyand analyzed by confocal microscopy. Upper, immunofluorescence.Lower, bright-field images

Fig. 3 The prediction ofEhRabB ubiquitination. TheEhRabB amino acid sequencewas analyzed using thebioinformaticsCKSAAP_UbSite web server(http://protein.cau.edu.cn/cksaap_ubsite/index.php) topredict ubiquitination potentialsites in this protein. Theprogram identified two lysineswith high prediction score. Thelysine residue position 189 hasa low grade of confidence(90 % specificity), and thelysine 183 has a high grade ofconfidence (98 % specificity)for Ub modification

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previously described (Rodriguez et al. 2000), the EhRabBprotein is located in cytoplasmic vesicles in trophozoiteswithout interaction with RBCs, but the EhRabB-containingvesicles were translocated to plasma membrane and to

phagocytic mouths during phagocytosis (Fig. 2). In theseexperiments, we also observed a decrease of the immuno-labeling at 5 min of phagocytosis and a subsequent augmentof the label at posterior times of interaction (Fig. 2).

All these results indicate that the presence of RBCsnegatively regulates the expression of the EhRabB proteinat the initial times of phagocytosis and suggest that the half-life of this protein is short, at least during phagocytosis.

Prediction of lysine ubiquitination of EhRabB

In eukaryotes, proteins that are targeted for proteasome-mediated degradation are often modified by ubiquitination.Typically, a covalent isopeptide bond is formed between theC-terminal carboxyl group of ubiquitin (Ub) and the ε-aminogroup of a lysine residue on the substrate protein. After aprotein is modified with Ub, additional isopeptide bonds canform between the C-terminal of another incoming Ub and oneof the seven lysine residues of the Ub on the modified proteinto generate polyubiquitin chains (Maupin-Furlow 2012).Indeed, the genes encoding for the enzymes involved in theUb-modification pathway have been identified in E.

Fig. 4 The expression of EhrabB mRNA during phagocytosis. Thetrophozoites of E. histolytica were incubated at 37 °C with RBCs atdifferent times. Then, the expression of the mRNA of EhrabB wasanalyzed by real-time RT-PCR. In these assays, the amplification of theE. histolytica18S rRNA was used as normalizer. Values are expressedusing the delta-delta Ct method to derive the relative fold change.Asterisks indicate significant difference (p<0.05) compared to troph-ozoites that were not in contact with RBCs

Fig. 5 EhrabB mRNA stability.Trophozoites were incubatedwith 10 μg/ml of actinomycinD for 1 h at 37 °C and thenincubated at different times inthe absence (−eryt) or in thepresence of human erythrocytes(+eryt). Then, the expression ofthe mRNA of EhrabB wasanalyzed by semiquantitativeRT-PCR. As internal control,we analyzed the expression ofthe 18S rRNA under the sameconditions. a RT-PCR ofEhrabB in the absence and inthe presence of RBCs. b Half-life of EhrabB. The bandsobtained by RT-PCR at eachtime were analyzed bydensitometry and the mRNAlevels were expressed as apercentage of the RNA levelafter the addition ofactinomycin D (time 0). Thehalf-life (t1/2) was calculated forEhrabB mRNA in the presence(t1/2=20.2 min) or in theabsence of erythrocytes (t1/2=120 min)

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histolytica (Arya et al. 2012), Thus, to predict potential ubiq-uitination sites in EhRabB, we used the bioinformatic toolnamed CKSAAP_UbSite (Chen et al. 2011). This programidentified two lysines that could be target for Ubmodification,one with a low grade of confidence (K189) and another with ahigh grade of confidence (K183) (Fig. 3). Although the ubiq-uitination of EhRabB must to be demonstrated, this findingsuggests that, at early times of phagocytosis, the Ub modifi-cation of this GTPase is possibly triggered for its degradation.Interestingly, it has been reported that ubiquitination and theproteasomal degradation of Ypt7, a Rab GTPase of yeast, areneeded for the regulation of the membrane fusion of the yeastvacuole (Kleijnen et al. 2007). Thus, ubiquitin–proteasomeregulationmay be involved in the membrane fusion controlledby EhRabB. These findings might provide useful insights forstudying a possible mechanism of the ubiquitination pathwaysin E. histolytica.

Expression of the mRNA of EhrabB during phagocytosis

The decrease of the levels of the EhRabB protein may perhapscorrelate with a diminishing in the mRNA amount. To inves-tigate this possibility, we analyzed the levels of the EhrabBtranscript during phagocytosis by real-time RT-PCR. Theseassays showed that the amount of EhrabB mRNA was ap-proximately 80 and 70 %, with respect to the control withoutRBCs, after 5 and 10 min of phagocytosis, respectively

(Fig. 4). Interestingly, after 30 min of interaction with RBCs,the EhrabB transcript increased up to 170 % (Fig. 4). Theseresults suggest that the reduction in the amount of EhRabBprotein during phagocytosis is due in part to a decrease in thelevels of its mRNA, although the diminishing of EhRabB wasmainly due to events related with the degradation of theprotein. Nevertheless, the diminishing of the transcript levelcould be the result of a short half-life of the mRNA and/or to arepression of its transcription.

Half-life of the EhrabB mRNA during phagocytosis

To analyze the possibility that the stability of the EhrabBmRNA is playing a role in the decrease of the gene expressionduring phagocytosis, we determined its half-life in actinomy-cin D-treated trophozoites in the absence or in the presence ofRBCs. For this analysis, the amount of EhrabB mRNA wasmeasured from 0 to 12 h by semiquantitative RT-PCR assays.As internal control, we analyzed the level of the 18S rRNA inthe same conditions. Results showed that, in the absence ofRBCs, the EhrabB transcript diminishes gradually, and themRNA was detected up to 3 h after treatment (Fig. 5a).Interestingly, in the presence of RBCs, the signal dramaticallydecreased during phagocytosis, and the transcript was weaklydetected after 60 min of interaction with RBCs (Fig. 5a). Inboth conditions, the 18S rRNA showed no significant varia-tions at the analyzed times.

Fig. 6 The promoter activity of URE-1 of the EhrabB gene duringphagocytosis. a Schematic representation of the EhrabB promoterregion. The URE-1motif and others cis consensus sequences are indi-cated (boxes). Arrows indicate the transcription initiation sites ofEhrabB gene. b Left, schematic representation of the relevant featuresof the plasmids used. The plasmid used for promoter activity of theEhrabB gene contains 428 bp upstream of the first initiation transcrip-tion site and +96 nt downstream from that site. CAT chloramphenicolacetyltransferase reporter gene. 3′ACT 3′-flanking region of the actingene. Act 480 bp of the 5′-flanking region of actin gene, P PstI, HHindIII, B BamHI, X XhoI, E EcoRI, K KpnI. Right, CAT activities oftransfected trophozoites. Transfected trophozoites were incubated at

37 °C with RBCs at different times before to determine the CATactivities. Activities are relative to that obtained with the actin promot-er gene, used as a positive control. Each bar corresponds to the averageof CAT activities±SD representative of three independent experimentsperformed by duplicated. c EMSA. The URE1 motif from the EhrabBgene promoter was radioactively labeled and incubated with 30 μg ofnuclear extracts obtained from trophozoites at different times of phago-cytosis. Then, DNA-protein complexes were analyzed by electropho-resis. Lane 1, free probe. Lanes 2, 5, 8, and 11, no competitor. Lanes 3,6, 9, and 12 specific competitor (SC) (150-fold excess of the same coldfragment). Lanes 4, 7, 10, and 13, unspecific competitor (UC) (150-fold excess of a mingled oligonucleotide with similar composition)

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The EhrabB mRNA and 18S rRNA were quantified bydensitometry and expressed in pixels. Then, to determine themRNA half-life, the pixels of the RT-PCR products wereplotted in a semilogarithmic scale against time (Fig. 5b). Inthese calculations, the amount of EhrabB and 18S rRNAimmediately after the actinomycin D treatment (t0) was takenas 100 %. In the absence of RBCs, the EhrabB mRNA half-life was estimated in 2 h, whereas in the presence of RBCs itwas shortened to 20 min (Fig. 4b), confirming a significantvariation in the decay rate of the EhrabB mRNAwhen troph-ozoites are in contact with target cells. On the other hand,these experiments showed that 18S rRNA decay remainedwith minimal changes during the 6-h course of the transcrip-tion inhibition in both conditions (Fig. 5b).

In eukaryotes, the stability of mRNA is mainly controlledby RNA interference processes (e.g., siRNA, microRNA) and

RNA-binding proteins, which act to selectively degrade orstabilize mRNAs. In mammals, a common feature of manyunstable mRNAs is the presence of adenylate–uridylate-richelements within the 3′-UTR (DeMaria and Brewer 1996). Thenucleotide sequence downstream to the stop codon of theEhrabB gene is rich in As and Us. However, most of the E.histolytica genes contain AU-rich sequences in their 3′-UTRs(Zamorano et al. 2008), and further experiments are needed toidentify destabilizing motifs in the mRNA of EhrabB.

Promoter assays during phagocytosis

To analyze also a possible decrease in the transcription ofthe EhRabB gene during phagocytosis, we analyzed theCAT activity in trophozoites transiently transfected withthe construct pRab428, which in functional assays showed

Fig. 7 The immunolocalizationof EhURE1BP duringphagocytosis. Trophozoiteswere grown in coverslides andincubated at 37 °C with RBCsat different times. Then,samples were fixed,permeabilized, and reacted withanti-EhURE-1BP antibody.Bound antibody was detectedwith a FITC-conjugatedsecondary antibody (green) andnuclei were counterstained withDAPI (blue). Finally, sampleswere analyzed by confocalmicroscopy. a, c, e, g Brightfield. b, d, f,h Immunofluorescence. Insetsshowed a magnification of thenuclei signaled. White dotsshowed co-localization

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the maximum promoter activity of EhrabB (Romero-Diaz etal. 2007). These experiments showed that transfected troph-ozoites displayed a 20 % reduction of CAT activity in thepresence of RBCs with respect to transfected trophozoites inthe absence of RBCs (Fig. 6b). These results suggest that,under our experimental conditions, the cis-activating do-main URE1 has an active role during phagocytosis. We alsoanalyzed the CAT activity during the phagocytosis of otherconstructs containing the different lengths of the EhRabBpromoter (Romero-Diaz et al. 2007). None of these con-structs showed significant participation in EhrabB genetranscription during phagocytosis (data not shown).

To confirm the role of the URE1 motif in the expression ofEhrabB during phagocytosis, we carried out electrophoreticmobility shift assay (EMSA) using nuclear extracts isolatedfrom trophozoites incubated with RBCs at different times. Inthe absence of RBCs, two DNA–protein complexes weredetected in these assays (Fig. 6c). The intensity of thesecomplexes was drastically diminished in the assays usingnuclear extracts from trophozoites after 5min of phagocytosis,and at posterior times, the intensity of these complexes waspartially recovered (Fig. 6c). In all cases, both complexes werecompeted by a 150-fold excess of the same cold sequence, butthey were not competed by a 350-fold excess of a nonspecificcompetitor (Fig. 6c), indicating the specificity of the DNA–protein binding. These results confirm that URE1 is involvedin the EhrabB expression during phagocytosis.

Location of EhURE1BP during phagocytosis

Recently, we identified the transcription factor that binds tothe cis-regulatory element URE1 (Calixto-Galvez et al.2011). To analyze the location of EhURE1BP throughphagocytosis, we carried out the immunolabeling of thisprotein in trophozoites during this event. As previouslydescribed (Calixto-Galvez et al. 2011), EhURE1BP wasdetected in dots inside the nuclei and into the cytoplasm(Fig. 7b). Interestingly, after 5 and 10 min of phagocytosis,when we observed a decrease in the amount of EhrabBtranscript, the presence of EhURE1BP in nuclei diminishedconsiderably with respect to time 0 (Fig. 7d, f), and after30 min of interaction with RBCs, which corresponds to thetime when we detected an increase in the mRNA level, thesignal detected inside the nuclei was higher to that observedin the absence of RBCs (Fig. 7h). These results suggest thatthe expression of the EhrabB gene is influenced by theability of EhURE1BP to be located inside the nucleus toachieve its function of transcription factor.

In conclusion, in this work, we showed that the expressionof the EhRabB protein is modified through phagocytosis; atearly times, its expression is diminished, but protein level wasrecovered at subsequent times. The diminishing of proteinexpression in the early times of phagocytosis is due in part

to a decrease in the mRNA level. The transcript reduction isresult of both a drastic reduction in its half-life and a diminish-ing in its transcription. In addition, we showed that the tran-scription of the EhrabB gene is determined, at least in part, bythe presence of EhURE1BP in the nucleus. Further experi-ments are required to determine the signaling pathways in-volved in the protein and mRNA degradation and in thetranslocation of EhURE1BP from nuclei to cytoplasm at theearly times of phagocytosis.

Acknowledgments This work was supported by Consejo Nacionalde Ciencia y Tecnología (CONACYT, Mexico). We thank Iván J.Galván for his invaluable technical assistance.

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