Parasitol Res (2013) 112:1631–1640 DOI 10.1007/s00436-013-3318-2
ORIGINAL PAPER
The small GTPase EhRabB of Entamoeba histolytica is 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 of Entamoeba histolytica is influenced by environmental conditions and that transcription profile changes occur during invasion, suggesting that gene expression may be involved in the virulence of this parasite. However, the molecular mechanisms that are implicated in the control of gene expression in this microorganism are poorly understood. Here, we showed that the expression of the EhRabB protein, a small GTPase involved in phagocytosis, is modified through the interaction with red blood cells. By ELISA, Western blot, and immunofluorescence assays, we observed that the expression of EhRabB diminished after 5 min of the interaction of trophozoites with red blood cells, but protein level was recovered at subsequent times. In the EhRabB amino acid sequence, we found two lysine residues that could be target for ubiquitin modification and trigger the degradation of this GTPase at early times of phagocytosis. The analysis of the expression of the EhrabB mRNA showed that the interaction of trophozoites with red blood cells produces a M. Hernandes-Alejandro : M. Calixto-Gálvez : A. Salas-Casas : J. Cázares-Ápatiga : E. Orozco : M. A. Rodríguez (*) Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, A.P. 14-740, 07000, Mexico, D.F., Mexico e-mail:
[email protected] I. López-Reyes Centro de Diagnóstico y Vigilancia Epidemiológica del Distrito Federal, Instituto de Ciencia y Tecnología del Distrito Federal, 06010, Mexico, D.F., Mexico Present Address: A. Salas-Casas Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Carretera Actopan-Tilcuautla, Exhacienda la Concepción S/N, San Agustin Tlaxiaca, Hidalgo, Mexico
drastic diminishing in its half-life. In addition, promoter assays using the chloramphenicol acetyltransferase reporter gene and electrophoretic mobility shift assays experiments showed that the URE1 motif located in the promoter region of EhrabB is involved in the expression regulation of this gene during phagocytosis. Moreover, the immunolocalization of the URE1-binding protein during phagocytosis indicated that the transcription of the EhrabB gene is determined, at least in part, by the translocation of this transcription factor to nuclei. These results suggested that the expression of particular genes of this parasite is controlled at several stages.
Introduction For all cells, the regulation of gene expression is a fundamental mechanism to drive development, homeostasis, and adaptation to the environment. In eukaryotes, any step of gene expression may be controlled, from the transcription of DNA into RNA to the post-translational modifications of the proteins. Transcription initiation is mediated by the cis-regulatory elements located in gene promoters, the concerted action of transcription factors along with the RNA polymerase II transcriptional machinery, and a diversity of co-regulators that bridge the DNA-binding factors to the transcriptional machinery (Hirose and Manley 2000). Then, post-transcriptional events such as mRNA processing and nuclear export, mRNA stability, and microRNA-dependent modulation create a complex intracellular network contributing to determine the global levels of specific mRNAs (Moore 2005). Most mRNA regulatory elements are located within the 5′ and 3′ untranslated regions (UTRs), where they function as platforms for the binding of numerous proteins and non-coding RNAs. The 5′UTR is principally involved in controlling mRNA translation
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(Pickering and Willis 2005), while the 3′-UTR regulates the multiple steps of mRNA metabolism and stability (Andreassi and Riccio 2009; Moore 2005). In addition, the covalent modification of proteins by ubiquitin and ubiquitin-like proteins is involved in the regulation of numerous cellular pathways cells (Kravtsova-Ivantsiv and Ciechanover 2011). In many cases, the modification is followed by the targeting of the tagged proteins to proteasomal or lysosomal degradation, thus terminating their function (Kravtsova-Ivantsiv and Ciechanover 2011). In particular, parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, or immune evasion, and the expression of these genes could be modulated by the target cell–parasite interaction or by some compounds present in the host tissues. For instance, bile induces the excystation of Entamoeba invadens cysts (Mitra et al. 2010) and also stimulates the expressions of genes that produce the energy required by newly excysted juvenile Clonorchis sinensis to migrate to the bile duct and to modulate the regulatory signals of cell proliferation associated with adult development (Kim et al. 2008). On the other hand, the release of reactive oxygen and nitrogen species, as part of the hosts’ innate immune response against infections, induces the expression of several parasite genes that are needed to survive into their hosts and contributes to disease outcome (Kim et al. 2012; Rastew et al. 2012). The protozoan Entamoeba histolytica is the etiologic agent of human amebiasis, a disease that affects up to 50 million people worldwide each year and causes from 40,000 to 100,000 deaths annually (WHO 1997). The molecular mechanisms participating in the parasite invasiveness are not completely understood, but it has been reported that the pathogenicity is influenced by environmental conditions such as bacteria flora and cholesterol (Bracha and Mirelman 1984; Meerovitch and Ghadirian 1978). Moreover, the transcription changes of several genes occur during invasion (Bruchhaus et al. 2002; Gilchrist et al. 2006; Gonzalez et al. 2011). Thus, modification in gene expression may be involved in the virulence of E. histolytica. However, despite some studies that have been performed to characterize the gene expression regulation of a few genes (Gomez et al. 2010), the molecular mechanisms that are implicated in the control of the gene expression in this parasite are poorly understood. EhRabB is a Rab GTPase of E. histolytica located close to plasma membrane and in phagocytic mouths when trophozoites are incubated with red blood cells (RBCs) (Rodriguez et al. 2000), suggesting that this protein is involved in phagocytosis, an event related to the virulence of the parasite (Orozco et al. 1983). Functional assays showed that the transcription of EhrabB is coordinately regulated by different cis-elements that are situated in its gene promoter.
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In basal conditions, the transcription of the EhrabB gene is regulated positively and negatively by two cis-elements (Romero-Diaz et al. 2007). An URE1 sequence situated between positions −428 and −413 with respect to the first initiation transcription site activates the gene transcription (Romero-Diaz et al. 2007), and recently, we identified a polypeptide containing staphyloccocal nuclease and Tudor domains as the URE1-binding protein (EhURE1BP) (Calixto-Galvez et al. 2011). On the other hand, the element that negatively controls the transcription of the EhrabB gene was situated in a fragment of 67 bp, between positions −491 and −428 (Romero-Diaz et al. 2007). In addition, the expression of EhrabB is modified by some environmental stimuli. Functional assays with a construct that includes seven putative heat shock elements of the EhrabB gene promoter showed an increase of twice the chloramphenicol acetyltransferase (CAT) activity of heat shocked trophozoites with respect to parasites maintained at 37 °C (Romero-Diaz et al. 2007). In concordance, the EhrabB transcript showed a significant augment in trophozoites submitted to heat shock (Romero-Diaz et al. 2007). Additionally, immunofluorescence assays showed an apparent variation in the amount of the EhRabB protein at different 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 early times of the phagocytosis of RBCs. The decrease in the levels of the EhrabB transcript during phagocytosis seems to be a consequence of a decrease in the mRNA half-life. In addition, we hypothesized that there are also a diminution in the transcription of EhrabB due to a reduction in the amount of 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-33 medium 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 incubation with distilled water. Then, the total extracts of trophozoites were obtained in the presence of protease inhibitors (Complete® Mini; Roche, Mannheim). At time 0, we used trophozoites 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 phagocytosis were incubated for 2 h at room temperature with specific antibodies against EhRabB (1:2,000) (Rodriguez et al. 2000). Then, the plates were incubated for 1 h at 37 °C with a mouse anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:10,000; Invitrogen). The reaction was 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 trophozoites were separated by 12 % SDS-PAGE and transferred to nitrocellulose filters. Then, membranes were incubated with antibodies against EhRabB (1:2,000), followed by incubation with a mouse anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:10,000; Invitrogen). Finally, the antibody detection was developed by chemiluminescence (ECL, GE Healthcare). As internal control, the same membranes were probed with antibodies against actin. Relative intensities were documented and analyzed by densitometry. For immunofluorescence, trophozoites grown on coverslides were incubated at 37 °C during different times with human RBCs (1:100). Then, samples were fixed with 4 % paraformaldehyde and permeabilized with 0.5 % Triton X100 for 30 min at room temperature. Samples were then incubated with 1 % BSA for 1 h at room temperature and incubated overnight at 4 °C with antibodies against EhRabB (1:500) or against EhURE1BP (1:6,000) (Calixto-Galvez et al. 2011). Then, cells were incubated for 1 h at 37 °C with a fluorescein-labeled secondary antibody (1:1,000; Zymed). Nuclei were counterstained with 30 nm of 4′,6-diamino-2phenilindole (DAPI) for 5 min. Samples were mounted with medium for fluorescence (VECTASHIELD; Vector Laboratories) and examined through a confocal microscope (Leica TCS SP2). Observations were performed in14 planes from the top to the bottom of each sample, and the distance between scanning planes was 1 μm. Real-time RT-PCR To evaluate the amount of the mRNA of EhrabB during phagocytosis, total RNA was obtained from trophozoites at different times of phagocytosis using the TRIzol Reagent (Gibco BRL) according to the manufacturer’s recommendations. Then, cDNA was synthesized using an oligo(dT) primer (Invitrogen), and PCR was performed utilizing specific primers for EhrabB and 18S rRNA (used as normalizer) (Romero-Diaz et al. 2007). Quantitative amplifications (qRT-PCR) were performed in the 7300/7500/7500 Fast Real-Time PCR System (Applied Biosystems) using the SYBR Green PCR Master Mix kit (Applied Biosystems) under the conditions previously described (Romero-Diaz et
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al. 2007). The two replicates of each assay were analyzed in triplicate. Relative quantification was performed using the delta–delta Ct method (Livak and Schmittgen 2001). EhrabB mRNA stability assays The half-life of the EhrabB transcript was determined as described (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 of 10 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 cDNA synthesis and reverse transcription (RT)-PCR were performed as described above. EhrabB mRNA levels were normalized with respect to the 18 rRNA amount. In these estimations, the EhrabB mRNA quantity from trophozoites in the absence of RBCs was taken as 100 %. Transient transfection and CAT assays To analyze the promoter activity during phagocytosis, we used the pRab428 construction, which displayed the greatest CAT activity on functional assays and contains the transcriptional activator URE1 (Romero-Diaz et al. 2007). As positive control, we used the pA5′A3′CAT vector that contains the E. histolytica actin gene promoter (Nickel and Tannich 1994) and as negative control the reporter vector without promoter region (pBSCAT-ACT). The transfection of trophozoites was carried out by electroporation as described (Nickel and Tannich 1994). CAT activity was determined by two-phase diffusion assays as described (Gomez et al. 1998), incubating 100 μg of extracts from trophozoites and 1.25 mM chloramphenicol with [14C]-acetyl CoA (PerkinElmer) within 4 h. The background activity displayed by trophozoites transfected with the promoter-less vector was subtracted from activity obtained from cells transfected with each construct. Activities were expressed as relative activity with respect to that obtained from trophozoites transfected with the plasmid pA5′A3′CAT. The enzymatic activity for each construct was assayed at least three times by duplicate. Electrophoretic mobility shift assays A double-stranded oligonucleotide corresponding to position −428 to −412 (5′-TCTTAAATATTTGGAAC-3′) of EhrabB and containing the URE1 motif (Romero-Diaz et al. 2007) was labeled at its 5′ end with T4 polynucleotide kinase (Invitrogen) and [γ-32P]ATP following standard procedures (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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The way by which host cells are killed and phagocytosed by E. histolytica follows a sequential model of adherence, cell killing, an initiation of phagocytosis, and engulfment, and several molecules are involved in these events (Sateriale and Huston 2011). Vesicular trafficking plays an essential role in the expression of virulence of this parasite, and this process is orchestrated by small GTPases, Rab proteins, which act as molecular switches regulating the fusion of vesicles with target membranes through the conformational change
between active (GTP-bound) and inactive (GDP-bound) forms (Stenmark and Olkkonen 2001). Thus, Rab proteins have an important role in the virulence of E. histolytica (Nozaki and Nakada-Tsukui 2006). EhRabB is a Rab GTPase of E. histolytica that is translocated to plasma membrane and phagocytic mouths during phagocytosis, and interestingly, the amount of EhRabB seems to be variable during the engulfment of human erythrocytes (Rodriguez et al. 2000). To confirm this finding, we analyzed the protein level of EhRabB during the phagocytosis of RBCs using specific antibodies against this protein (Rodriguez et al. 2000). The data obtained from trophozoites in the absence of RBCs (time 0) were arbitrarily taken as 100 % of expression. In all experiments, antibodies against actin were used as internal control. By ELISA, we observed only about 20 % of the EhRabB expression at 5 min of phagocytosis (Fig. 1a). After this time, the quantity of EhRabB was sequentially recovered, and after 30 min of phagocytosis, we detected approximately 80 % of expression with respect to that observed in the absence of RBCs (Fig. 1a). In contrast, the expression of actin was similar during the different times of phagocytosis (Fig. 1a), suggesting that the EhRabB expression varies during phagocytosis. These observations were corroborated by Western blot assays. In these experiments, the intensity of the 21-kDa band recognized by the antibodies against EhRabB decreased after 5 min of interaction with RBCs, but the signal of this band enhanced at subsequent times of phagocytosis reaching almost the same level as that of the control trophozoites after 30 min of phagocytosis (Fig. 1b). On the
Fig. 1 The expression of EhRabB protein during the phagocytosis of RBCs. The trophozoites of E. histolytica were incubated at 37 °C with RBCs at different times. Then, the expression of EhRabB was analyzed using specific antibodies against it. As internal control, we used antibodies against actin to analyze the expression of this protein under the same conditions. a ELISA. b Western blot. c The relative expression of
EhRabB. The relative intensities of the bands detected by Western blot were documented and analyzed by densitometry. The protein level of EhRabB was normalized to the actin protein level of each sample, and the value obtained in the absence of RBCs (time 0) was taken arbitrarily as 1. Bars show the mean levels (±SD). Asterisks indicate significant difference (p
