EhVps23, an ESCRT-I Member, Is a Key Factor in Secretion, Motility, Phagocytosis and Tissue Invasion by Entamoeba histolytica.

The EhVps23 protein, an orthologue of the yeast Vps23 and the mammalian TSG101 proteins, is the single member of the ESCRT-I complex of Entamoeba histolytica identified and characterized until now. EhVps23 actively participates in vesicular trafficking and phagocytosis, which influence several cellular events. In this paper, we investigated the role of EhVps23 in virulence-related functions, including the invasive capacity of trophozoites, using transfected trophozoites. Trophozoites overexpressing the EhVps23 protein (Neo-EhVps23) presented helical arrangements in the cytoplasm, similar to the ones formed by EhVps32 for scission of vesicles. By confocal and transmission electron microscopy, EhVps23 was detected in multivesicular bodies, vesicles, and the extracellular space. It was secreted in vesicles together with other proteins, including the EhADH adhesin. Probably, these vesicles carry molecules that participate in the prey capture or in cell-cell communication. Mass spectrometry of precipitates obtained using α-EhVps23 antibodies, evidenced the presence of proteins involved in motility, phagocytosis, vesicular trafficking and secretion. The study of cellular functions, revealed that Neo-EhVps23 trophozoites exhibit characteristics similar to those described for mammalian transformed cells: they grew 50% faster than the control; presented a significant higher rate of phagocytosis, and migrated five-fold faster than the control, in concordance with the low rate of migration exhibited by Ehvps23-knocked down trophozoites. In addition, Neo-EhVps23 trophozoites produced dramatic liver abscesses in experimental animals. In conclusion, our results showed that EhVps23 overexpression gave to the trophozoites characteristics that resemble cancer cells, such as increased cell proliferation, migration, and invasion. The mutant that overexpresses EhVps23 can be a good study model to explore different events related to the transformation of malignant cells.

Host Invasion by Pathogenic Amoebae: Epithelial Disruption by Parasite Proteins.

The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleriafowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.

Epithelial Cells Expressing EhADH, An Entamoeba histolytica Adhesin, Exhibit Increased Tight Junction Proteins.

In Entamoeba histolytica, the EhADH adhesin together with the EhCP112 cysteine protease, form a 124 kDa complex named EhCPADH. This complex participates in trophozoite adherence, phagocytosis and cytolysis of target cells. EhCPADH and EhCP112 are both involved on epithelium damage, by opening tight junctions (TJ) and reaching other intercellular junctions. EhADH is a scaffold protein belonging to the ALIX family that contains a Bro1 domain, expresses at plasma membrane, endosomes and cytoplasm of trophozoites, and is also secreted to the medium. Contribution of EhADH to TJ opening still remains unknown. In this paper, to elucidate the role of EhADH on epithelium injury, we followed two strategies: producing a recombinant protein (rEhADH) and transfecting the ehadh gene in MDCK cells. Results from the first strategy revealed that rEhADH reached the intercellular space of epithelial cells and co-localized with claudin-1 and occludin at TJ region; later, rEhADH was mainly internalized by clathrin-coated vesicles. In the second strategy, MDCK cells expressing EhADH (MDCK-EhADH) showed the adhesin at plasma membrane. In addition, MDCK-EHADH cells exhibited adhesive features, producing epithelial aggregation and adherence to erythrocytes, as described in trophozoites. Surprisingly, the adhesin expression produced an increase of claudin-1, occludin, ZO-1 and ZO-2 at TJ, and also the transepithelial electric resistance (TEER), which is a measure of TJ gate function. Moreover, MDCK-EhADH cells resulted more susceptible to trophozoites attack, as showed by TEER and cytopathic experiments. Overall, our results indicated that EhADH disturbed TJ from the extracellular space and also intracellularly, suggesting that EhADH affects by itself TJ proteins, and possibly synergizes the action of other parasite molecules during epithelial invasion.

A Calcium/Cation Exchanger Participates in the Programmed Cell Death and in vitro Virulence of Entamoeba histolytica.

Entamoeba histolytica is the etiologic agent of human amoebiasis, disease that causes 40,000 to 100,000 deaths annually worldwide. The cytopathic activity as well as the growth and differentiation of this microorganism is dependent on both, extracellular and free cytoplasmic calcium. However, few is known about the proteins that regulate the calcium flux in this parasite. In many cells, the calcium extrusion from the cytosol is performed by plasma membrane Ca2+-ATPases and calcium/cation exchangers. The aim of this work was to identify a calcium/cation exchanger of E. histolytica and to analyze its possible role in some cellular processes triggered by calcium flux, such as the programmed cell death and in vitro virulence. By searching putative calcium/cation exchangers in the genome database of E. histolyica we identified a protein belonging to the CCX family (EhCCX). We generated a specific antibody against EhCCX, which showed that this protein was expressed in higher levels in E. histolytica than its orthologous in the non-pathogenic amoeba E. dispar. In addition, the expression of EhCCX was increased in trophozoites incubated with hydrogen peroxide. This E. histolytica exchanger was localized in the plasma membrane and in the membrane of some cytoplasmic vesicles. However, after 10 min of erythrophagocytosis, EhCCX was found predominantly in the plasma membrane of the trophozoites. On the other hand, the parasites that overexpress this exchanger contained higher cytosolic calcium levels than control, but the extrusion of calcium after the addition of hydrogen peroxide was more efficient in EhCCX-overexpressing trophozoites; consequently, the programmed cell death was retarded in these parasites. Interestingly, the overexpression of EhCCX increased the in vitro virulence of trophozoites. These results suggest that EhCCX plays important roles in the programmed cell death and in the in vitro virulence of E. histolytica.

Entamoeba histolytica EhCP112 Dislocates and Degrades Claudin-1 and Claudin-2 at Tight Junctions of the Intestinal Epithelium.

During intestinal invasion, Entamoeba histolytica opens tight junctions (TJs) reflected by transepithelial electrical resistance (TEER) dropping. To explore the molecular mechanisms underlying this, we studied in vitro and in vivo the damage produced by the recombinant E. histolytica cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional in vitro assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. In vivo, rEhCP112 increased intestinal epithelial permeability in the mouse colon, likely due to apical erosion and claudin-1 and claudin-2 degradation. In conclusion, we provide evidence that EhCP112 causes epithelial dysfunction by specifically altering claudins at TJ. Thus, EhCP112 could be a potential target for therapeutic approaches against amoebiasis.

Adherens junctions and desmosomes are damaged by Entamoeba histolytica: Participation of EhCPADH complex and EhCP112 protease.

Entamoeba histolytica trophozoites adhere to epithelium at the cell-cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded ß-cat, E-cad, Dsp l/ll, and Dsg-2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with ß-cat, E-cad, Dsp l/ll, and Dsg-2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin-coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with ß-cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E-cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.

Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein-protein docking.

EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein-protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442-479 residues, adjacent to the target cell-adherence region (480-600 residues), and by the Bro1 domain (9-349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.

Multinucleation and Polykaryon Formation is Promoted by the EhPC4 Transcription Factor in Entamoeba histolytica.

Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica.

The knockdown of each component of the cysteine proteinase-adhesin complex of Entamoeba histolytica (EhCPADH) affects the expression of the other complex element as well as the in vitro and in vivo virulence.

Entamoeba histolytica is the protozoan parasite causative of human amoebiasis, disease responsible for 40 000-100 000 deaths annually. The cysteine proteinase-adhesin complex of this parasite (EhCPADH) is a heterodimeric protein formed by a cysteine protease (EhCP112) and an adhesin (EhADH) that plays an important role in the cytopathic mechanism of this parasite. The coding genes for EhCP112 and EhADH are adjacent in the E. histolytica genome, suggesting that their expression may be co-regulated, but this hypothesis has not yet been confirmed. Here, we performed the knockdown of EhCP112 and EhADH using gene-specific short-hairpin RNAs (shRNA), and the effect of these knockdowns on the expression of both complex components as well as on the in vitro and in vivo virulence was analysed. Results showed that the knockdown of one of the EhCPADH components produced a simultaneous downregulation of the other protein. Accordingly, a concomitant reduction in the overall expression of the complex was observed. The downregulation of each component also produced a significant decrease in the in vitro and in vivo virulence of trophozoites. These results demonstrated that the expression of EhCP112 and EhADH is co-regulated and confirmed that the EhCPADH complex plays an important role in E. histolytica virulence.

Analysis of the epithelial damage produced by Entamoeba histolytica infection.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

A recombinant influenza virus vaccine expressing the F protein of respiratory syncytial virus.

Infections with influenza and respiratory syncytial virus (RSV) rank high among the most common human respiratory diseases worldwide. Previously, we developed a replication-incompetent influenza virus by replacing the coding sequence of the PB2 gene, which encodes one of the viral RNA polymerase subunits, with that of a reporter gene. Here, we generated a PB2-knockout recombinant influenza virus expressing the F protein of RSV (PB2-RSVF virus) and tested its potential as a bivalent vaccine. In mice intranasally immunized with the PB2-RSVF virus, we detected high levels of antibodies against influenza virus, but not RSV. PB2-RSVF virus-immunized mice were protected from a lethal challenge with influenza virus but experienced severe body weight loss when challenged with RSV, indicating that PB2-RSVF vaccination enhanced RSV-associated disease. These results highlight one of the difficulties of developing an effective bivalent vaccine against influenza virus and RSV infections.

Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis in humans, affecting up to 50 million people worldwide each year and causing 40,000 to 100,000 deaths annually. EhCP112 is a cysteine proteinase of E. histolytica able to disrupt cell monolayers and digest extracellular matrix proteins, it is secreted by trophozoites and it can be active in a wide range of temperature and pH. These characteristics have encouraged the use of EhCP112 in the design and production of possible vaccines against amoebiasis, obtaining promising results. Nevertheless, we have no conclusive information about the role of EhCP112 in the E. histolytica pathogenesis. METHODS: A set of three specific siRNA sequences were used to silence the Ehcp112 gene via the soaking system. Silencing was evaluated by Western blot using an antibody against the EhCP112 recombinant protein. Finally, we analyzed the protease activity, the phagocytosis rate and the ability to destroy MDCK cells of the EhCP112-silenced trophozoites. RESULTS: The highest silencing effect on EhCP112 was detected at 16 h of treatment; time enough to perform the in vitro virulence assays, which showed that EhCP112 silencing produces a significant reduction in cytolysis and phagocytosis of target cells, indicating the participation of this proteinase in these events. CONCLUSIONS: EhCP112 is involved in the in vitro virulence of E. histolytica.

The EhCPADH112 complex of Entamoeba histolytica interacts with tight junction proteins occludin and claudin-1 to produce epithelial damage.

Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction.

Identification of a polypeptide containing Tudor and staphyloccocal nuclease-like domains as the sequence-specific binding protein to the upstream regulatory element 1 of Entamoeba histolytica.

Transcription initiation is the most regulated stage for the control of gene expression. This event requires that a complex of proteins called transcription factors bind to DNA through cis-regulatory elements located in the gene promoters. However, little is known about transcription regulation in Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis. Some genes encoding for proteins involved in the parasite pathogenicity contain specific upstream regulatory elements (URE1-URE5) in their promoters. Here, we identified the protein that specifically binds to the URE1 sequence (EhURE1BP). This protein contains five SNase domains and one Tudor motif, and has 21% identity and 36% similarity to the multifunctional eukaryotic protein known as the protein containing Tudor and staphyloccocal nuclease-like domains (TSN). To obtain antibodies against EhURE1BP, the recombinant protein was expressed and immunised in rabbits. Western blot and immunofluorescence assays showed that EhURE1BP is located in both nuclei and cytoplasm. Electrophoretic mobility shift assays and supershift assays demonstrated that EhURE1PB specifically binds to URE1 and that the C-terminus that includes the Tudor motif contains the DNA-binding domain of this protein. Results suggest that this TSN-like protein is the transcription factor that activates the transcription of some pathogenicity-related genes of E. histolytica.

Identification of four Entamoeba histolytica organellar DNA polymerases of the family B and cellular localization of the Ehodp1 gene and EhODP1 protein.

We report the identification of a family of four active genes (Ehodp1, Ehodp2, Ehodp3, and Ehodp4) encoding putative DNA polymerases in Entamoeba histolytica, the protozoan parasite responsible of human amoebiasis. The four Ehodp genes show similarity to DNA polymerases encoded in fungi and plant mitochondrial plasmids. EhODP polypeptides conserve the 3'-5' exonuclease II and 5'-3' polymerization domains, and they have the I, II, and III conserved boxes that characterize them as DNA polymerases of family B. Furthermore, we found in EhODP polymerases two novel A and B boxes, present also in DNA polymerases encoded in fungi mitochondrial plasmids. By in situ PCR, Ehodp1 gene was located in nuclei and in DNA-containing cytoplasmic structures. Additionally, using polyclonal antibodies against a recombinant rEhODP1-168 polypeptide, and confocal microscopy, EhODP1 was located in cytoplasmic DNA-containing structures.

The R2R3 Myb protein family in Entamoeba histolytica.

The MYB DNA-binding domain is conserved in vertebrates, plants, and fungi. This domain mediates the DNA-binding activity of proteins (that have transcription factor activity) in a sequence-specific manner and is also used for the protection of telomeric regions. The MYB DNA-binding domain contains three imperfect conserved repeats of 52 amino acids (R1, R2, and R3). Within each repeat, there are three tryptophans that are separated by 18 or 19 amino acids. In order to understand the role of Myb transcription factors in Entamoeba histolytica, we searched for MYB DNA-binding domain containing proteins using the amino acid sequence of human c-Myb as the query. We found 34 putative MYB DNA-binding domain containing proteins, which clustered into three monophyletic groups. Family I members conserve only the R2 and R3 repeats in their MYB DNA-binding domain and were dubbed in this report as EhMybR2R3. Family II includes single-repeat proteins related to human telomeric binding proteins. Family III is predicted to comprise proteins with one single repeat where the region corresponding to the conserved tryptophan of the third alpha helix is replaced by a (S)/(T)HAQK(Y)/(F)F motif; this family was named EhMybSHAQKYF. In this work, we focused on proteins that belong to the EhMybR2R3 family. RT-PCR analysis showed that EhMybR2R3 genes were differentially expressed in trophozoites grown in basal culture conditions. Purified rEhMyb10 protein, belonging to the EhMybR2R3 family, was able to bind a consensus Myb recognition element in vitro. In addition, using nuclear extracts from trophozoites of E. histolytica, we were able to detect Myb DNA-binding activity to this sequence. Our in silico surveys demonstrated that this consensus sequence is present in E. histolytica gene promoters. Interestingly, these promoters include different families of genes that are related to signal transduction, vesicular transport, heat shock response, and virulence. Thus, Myb putative transcription factors in E. histolytica could be involved in the transcriptional regulation of genes participating in several different pathways.

Entamoeba histolytica: a unicellular organism containing two active genes encoding for members of the TBP family.

Entamoeba histolytica is the protozoan parasite which causes human amoebiasis. In this parasite, few encoding genes for transcription factors have been cloned and characterized. The E. histolytica TATA-box binding protein (EhTBP) is the first basal transcription factor that has been studied. To continue with the identification of other members of the basal transcription machinery, we performed an in silico analysis of the E. histolytica genome and found three loci encoding for polypeptides with similarity to EhTBP. One locus has a 100% identity to the previously Ehtbp gene reported by our group. The second locus encodes for a 212 aa polypeptide that is 100% identical to residues 23-234 from EhTBP. The third one encodes for a 216 aa polypeptide of 24kDa that showed 42.6% identity and 73.7% similarity to EhTBP. This protein was named E. histolytica TBP-related factor 1 (EhTRF1). Ehtrf1 gene was expressed in bacteria and the purified 28kDa recombinant polypeptide showed the capacity to bind to TATTTAAA-box by electrophoretic mobility shift assays. K(D) values for rEhTBP and rEhTRF1 were (1.71+/-2.90)x10(-12)M and (1.12+/-0.160)x10(-11)M, respectively. Homology modeling of EhTRF1 and EhTBP revealed that, although they were very similar, they showed some differences on their surfaces. Thus, E. histolytica is a unicellular organism having two members of the TBP family.

A pcDNA-Ehcpadh vaccine against Entamoeba histolytica elicits a protective Th1-like response in hamster liver.

DNA vaccines are promising tools to fight parasitic diseases, including amoebiasis caused by the protozoan Entamoeba histolytica. Here we studied the immunogenicity and protective efficacy of a DNA vaccine against this parasite composed by the EhCPADH surface complex encoding genes (Ehcp112 and Ehadh112). EhCPADH is formed by an adhesin (EhADH112) and a cysteine proteinase (EhCP112), both involved in the parasite virulence. We evaluated transcription, protein expression, immunological response and protection against hepatic amoebiasis in hamsters intradermally and intramuscularly immunized with a mixture of pcDNA-Ehadh112 and pcDNA-Ehcp112 plasmids. RT-PCR and immunohistochemical assays showed that both antigens were differentially expressed in spleen and liver of immunized animals. No significant antibody immune response was induced by either route. However, intradermally inoculated hamsters presented a robust Th1-like immune response, characterized by high levels of INF-gamma and TNF-alpha cytokines, detected in the liver of animals challenged with virulent trophozoites. Animals showed significant protection against amoebiasis manifested by a higher survival rate and a significant prevention of liver abscess formation. We conclude that a refinement of this DNA vaccine could be a good choice to control hepatic amoebiasis.

Purification, refolding and autoactivation of the recombinant cysteine proteinase EhCP112 from Entamoeba histolytica.

The cysteine proteinase EhCP112 and the adhesin EhADH112 assemble to form the EhCPADH complex involved in Entamoeba histolytica virulence. To further characterize this cysteine proteinase, the recombinant full-length EhCP112 enzyme was expressed and purified under denaturing conditions. After a refolding step under reductive conditions, the inactive precursor (ppEhCP112) was processed to a 35.5 kDa mature and active enzyme (EhCP112). The thiol specific inhibitor E-64, but not serine or aspartic proteinase inhibitors arrested this activation process. The activation step of the proenzyme followed by the mature enzyme suggests an autocatalytic process during EhCP112 maturation. The experimentally determined processing sites observed during EhCP112 activation lie close to processing sites of other cysteine proteinases from parasites. The kinetic parameters of the mature EhCP112 were determined using hemoglobin and azocasein as substrates. The proteinase activity of EhCP112 was completely inhibited by thiol inhibitors, E-64, TLCK, and chymostatin, but not by general proteinase inhibitors. Since EhCP112 is a proteinase involved in the virulence of E. histolytica, a reliable source of active EhCP112 is a key step for its biochemical characterization and to carry out future protein structure-function studies.

The vacuolar ATPase from Entamoeba histolytica: molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein.

BACKGROUND: Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. RESULTS: We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. CONCLUSION: We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits were found in the E. histolytica genome, indicating the conserved nature of V-ATPase in this parasite.