Lysine Methyltransferase EhPKMT2 Is Involved in the In Vitro Virulence of Entamoeba histolytica.

Lysine methylation, a posttranslational modification catalyzed by protein lysine methyltransferases (PKMTs), is involved in epigenetics and several signaling pathways, including cell growth, cell migration and stress response, which in turn may participate in virulence of protozoa parasites. Entamoeba histolytica, the etiologic agent of human amebiasis, has four PKMTs (EhPKMT1 to EhPKMT4), but their role in parasite biology is unknown. Here, to obtain insight into the role of EhPKMT2, we analyzed its expression level and localization in trophozoites subjected to heat shock and during phagocytosis, two events that are related to amoeba virulence. Moreover, the effect of EhPKMT2 knockdown on those activities and on cell growth, migration and cytopathic effect was investigated. The results indicate that this enzyme participates in all these cellular events, suggesting that it could be a potential target for development of novel therapeutic strategies against amebiasis.

Dynamic Association of ESCRT-II Proteins with ESCRT-I and ESCRT-III Complexes during Phagocytosis of Entamoeba histolytica.

By their active movement and voraux phagocytosis, the trophozoites of Entamoeba histolytica constitute an excellent system to investigate the dynamics of the Endosomal Sorting Complex Required for Transport (ESCRT) protein interactions through phagocytosis. Here, we studied the proteins forming the E. histolytica ESCRT-II complex and their relationship with other phagocytosis-involved molecules. Bioinformatics analysis predicted that EhVps22, EhVps25, and EhVps36 are E. histolytica bona fide orthologues of the ESCRT-II protein families. Recombinant proteins and specific antibodies revealed that ESCRT-II proteins interact with each other, with other ESCRT proteins, and phagocytosis-involved molecules, such as the adhesin (EhADH). Laser confocal microscopy, pull-down assays, and mass spectrometry analysis disclosed that during phagocytosis, ESCRT-II accompanies the red blood cells (RBCs) from their attachment to the trophozoites until their arrival to multivesicular bodies (MVBs), changing their interactive patterns according to the time and place of the process. Knocked-down trophozoites in the Ehvps25 gene presented a 50% lower rate of phagocytosis than the controls and lower efficiency to adhere RBCs. In conclusion, ESCRT-II interacts with other molecules during prey contact and conduction throughout the phagocytic channel and trophozoites membranous system. ESCRT-II proteins are members of the protein chain during vesicle trafficking and are fundamental for the continuity and efficiency of phagocytosis.

Stepwise remodeling and subcompartment formation in individual vesicles by three ESCRT-III proteins.

The endosomal sorting complex required for transport (ESCRT) is a multi-protein machinery involved in several membrane remodeling processes. Different approaches have been used to resolve how ESCRT proteins scission membranes. However, the underlying mechanisms generating membrane deformations are still a matter of debate. Here, giant unilamellar vesicles, microfluidic technology, and micropipette aspiration are combined to continuously follow the ESCRT-III-mediated membrane remodeling on the single-vesicle level for the first time. With this approach, we identify different mechanisms by which a minimal set of three ESCRT-III proteins from Entamoeba histolytica reshape the membrane. These proteins modulate the membrane stiffness and spontaneous curvature to regulate bud size and generate intraluminal vesicles even in the absence of ATP. We demonstrate that the bud stability depends on the protein concentration and membrane tension. The approaches introduced here should open the road to diverse applications in synthetic biology for establishing artificial cells with several membrane compartments.

Molecular interplays of the Entamoeba histolytica endosomal sorting complexes required for transport during phagocytosis.

Entamoeba histolytica, the causative agent of human amoebiasis, exhibits a continuous membrane remodelling to exert its virulence properties. During this dynamic process, the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is a key player, particularly in phagocytosis, a virulence hallmark of this parasite. In addition to ESCRT, other molecules contribute to membrane remodelling, including the EhADH adhesin, EhRabs, actin, and the lysobisphosphatidic acid (LBPA). The endocytosis of a prey or molecules induces membrane invaginations, resulting in endosome and multivesicular bodies (MVBs) formation for cargo delivery into lysosomes. Alternatively, some proteins are recycled or secreted. Most of these pathways have been broadly characterized in other biological systems, but poorly described in protozoan parasites. Here, we encompass 10 years of ESCRT research in E. histolytica, highlighting the role of the ESCRT-I and ESCRT-III components and the EhADH and EhVps4-ATPase accessory proteins during phagocytosis. In particular, EhADH exhibits a multifunctional role along the endocytic pathway, from cargo recognition to endosome maturation and lysosomal degradation. Interestingly, the interaction of EhADH with EhVps32 seems to shape a concurrent route to the conventional one for MVBs biogenesis, that could optimize their formation. Furthermore, this adhesin is secreted, but its role in this event remains under study. Other components from the endosomal pathway, such as EhVps23 and LBPA, are also secreted. A proteomic approach performed here, using an anti-LBPA antibody, revealed that some proteins related to membrane trafficking, cellular transport, cytoskeleton dynamics, and transcriptional and translational functions are secreted and associated to LBPA. Altogether, the accumulated knowledge around the ESCRT machinery in E. histolytica, points it out as a dynamic platform facilitating the interaction of molecules participating in different cellular events. Seen as an integrated system, ESCRTs lead to a better understanding of E. histolytica phagocytosis.

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.

The novel EhHSTF7 transcription factor displays an oligomer state and recognizes a heat shock element in the Entamoeba histolytica parasite.

The heat shock response is a conserved mechanism that allows cells to respond and survive stress damage and is transcriptionally regulated by the heat shock factors and heat shock elements. The P-glycoprotein confer the multidrug resistance phenotype; Entamoeba histolytica has the largest multidrug resistance gene family described so far; one of these genes, the EhPgp5 gene, has an emetine-inducible expression. A functional heat shock element was localized in the EhPgp5 gene promoter, indicating transcriptional regulation by heat shock factors. In this work, we determined the oligomer state of EhHSTF7 and the recognition of the heat shock element of the EhPgp5 gene. The EhHSTF7 recombinant protein was obtained as monomer and oligomer. In silico molecular docking predicts protein-DNA binding between EhHSTF7 and 5'-GAA-3' complementary bases. The rEhHSTF7 protein specifically binds to the heat shock element of the EhPgp5 gene in gel shift assays. The competition assays with heat shock element mutants indicate that 5'-GAA-3' complementary bases are necessary for the rEhHSTF7 binding. Finally, the siRNA-mediated knockdown of Ehhstf7 expression causes downregulation of EhPgp5 expression, suggesting that EhHSTF7 is likely to play a key role in the E. histolytica multidrug resistance. This is the first report of a transcription factor that recognizes a heat shock element from a gene involved in drug resistance in parasites. However, further analysis needs to demonstrate the biological relevance of the EhHSTF7 and the rest of the heat shock factors of E. histolytica, to understand the underlying regulation of transcriptional control in response to stress.

Corrigendum: The sole DNA ligase in Entamoeba histolytica is a high-fidelity DNA ligase involved in DNA damage repair.

[This corrects the article DOI: 10.3389/fcimb.2018.00214.].

EhVps23: A Component of ESCRT-I That Participates in Vesicular Trafficking and Phagocytosis of Entamoeba histolytica.

The endosomal sorting complex required for transport (ESCRT) is formed by ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins. It conducts vesicular trafficking in eukaryotes through the formation of vesicles and membrane fission and fusion events. The trophozoites of Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents an active membrane movement in basal state that increases during phagocytosis and tissue invasion. ESCRT-III complex has a pivotal role during these events, but ESCRT-0, ESCRT-I and ESCRT-II have been poorly studied. Here, we unveiled the E. histolytica ESCRT-I complex and its implication in vesicular trafficking and phagocytosis, as well as the molecular relationships with other phagocytosis-involved molecules. We found a gene encoding for a putative EhVps23 protein with the ubiquitin-binding and Vps23 core domains. In basal state, it was in the plasma membrane, cytoplasmic vesicles and multivesicular bodies, whereas during phagocytosis it was extensively ubiquitinated and detected in phagosomes and connected vesicles. Docking analysis, immunoprecipitation assays and microscopy studies evidenced its interaction with EhUbiquitin, EhADH, EhVps32 proteins, and the lysobisphosphatidic acid phospholipid. The knocking down of the Ehvps23 gene resulted in lower rates of phagocytosis. Our results disclosed the concert of finely regulated molecules and vesicular structures participating in vesicular trafficking-related events with a pivotal role of EhVps23.

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites.

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

The atypical protein arginine methyltrasferase of Entamoeba histolytica (EhPRMTA) is involved in cell proliferation, heat shock response and in vitro virulence.

Protein arginine methylation regulates several cellular events, including epigenetics, splicing, translation, and stress response, among others. This posttranslational modification is catalyzed by protein arginine methyltransferases (PRMTs), which according to their products are classified from type I to type IV. The type I produces monomethyl arginine and asymmetric dimethyl arginine; in mammalian there are six families of this PRMT type (PRMT1, 2, 3, 4, 6, and 8). The protozoa parasite Entamoeba histolytica has four PRMTs related to type I; three of them are similar to PRMT1, but the other one does not show significant homology to be grouped in any known PRMT family, thus we called it as atypical PRMT (EhPRMTA). Here, we showed that EhPRMTA does not contain several of the canonical amino acid residues of type I PRMTs, confirming that it is an atypical PRMT. A specific antibody against EhPRMTA localized this protein in cytoplasm. The recombinant EhPRMTA displayed catalytic activity on commercial histones and the native enzyme modified its expression level during heat shock and erythrophagocytosis. Besides, the knockdown of EhPRMTA produced an increment in cell growth, and phagocytosis, but decreases cell migration and the survival of trophozoites submitted to heat shock, suggesting that this protein is involved in regulate negatively or positively these events, respectively. Thus, results suggest that this methyltransferase regulates some cellular functions related to virulence and cell surviving.

A noncanonical GATA transcription factor of Entamoeba histolytica modulates genes involved in phagocytosis.

In this paper, we explored the presence of GATA in Entamoeba histolytica and their function as regulators of phagocytosis-related genes. Bioinformatics analyses evidenced a single 579 bp sequence encoding for a protein (EhGATA), smaller than GATA factors of other organisms. EhGATA appeared phylogenetically close to Dictyostelium discoideum and Schistosoma mansoni GATA proteins. Its sequence predicts the presence of a zinc-finger DNA binding domain and an AT-Hook motif; it also has two nuclear localization signals. By transmission electron and confocal microscopy, anti-EhGATA antibodies revealed the protein in the cytoplasm and nucleus, and 65% of nuclear signal was in the heterochromatin. EhGATA recombinant protein and trophozoites nuclear extracts bound to GATA-DNA consensus sequence. By in silico scrutiny, 1,610 gene promoters containing GATA-binding sequences appeared, including Ehadh and Ehvps32 promoters, whose genes participate in phagocytosis. Chromatin immunoprecipitation assays showed that EhGATA interact with Ehadh and Ehvps32 promoters. In EhGATA-overexpressing trophozoites (NeoGATA), the Ehadh and Ehvps32 mRNAs amount was modified, strongly supporting that EhGATA could regulate their transcription. NeoGATA trophozoites exhibited rounded shapes, high proliferation rates, and diminished erythrophagocytosis. Our results provide new insights into the role of EhGATA as a noncanonical transcription factor, regulating genes associated with phagocytosis.

Lipids in Entamoeba histolytica: Host-Dependence and Virulence Factors.

Lipids are essential players in parasites pathogenesis. In particular, the highly phagocytic trophozoites of Entamoeba histolytica, the causative agent of amoebiasis, exhibit a dynamic membrane fusion and fission, in which lipids strongly participate; particularly during the overstated motility of the parasite to reach and attack the epithelia and ingest target cells. Synthesis and metabolism of lipids in this protozoan present remarkable difference with those performed by other eukaryotes. Here, we reviewed the current knowledge on lipids in E. histolytica. Trophozoites synthesize phosphatidylcholine and phosphatidylethanolamine by the Kennedy pathway; and sphingolipids, phosphatidylserine, and phosphatidylinositol, by processes similar to those used by other eukaryotes. However, trophozoites lack enzymes for cholesterol and fatty acids synthesis, which are scavenged from the host or culture medium by specific mechanisms. Cholesterol, a fundamental molecule for the expression of virulence, is transported from the medium into the trophozoites by EhNPC1 and EhNPC2 proteins. Inside cells, lipids are distributed by different pathways, including by the participation of the endosomal sorting complex required for transport (ESCRT), involved in vesicle fusion and fission. Cholesterol interacts with the phospholipid lysobisphosphatidic acid (LBPA) and EhADH, an ALIX family protein, also involved in phagocytosis. In this review, we summarize the known information on phospholipids synthesis and cholesterol transport as well as their metabolic pathways in E. histolytica; highlighting the mechanisms used by trophozoites to dispose lipids involved in the virulence processes.

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.

EhRabB mobilises the EhCPADH complex through the actin cytoskeleton during phagocytosis of Entamoeba histolytica.

Movement and phagocytosis are clue events in colonisation and invasion of tissues by Entamoeba histolytica, the protozoan causative of human amoebiasis. During phagocytosis, EhRab proteins interact with other functional molecules, conducting them to the precise cellular site. The gene encoding EhrabB is located in the complementary chain of the DNA fragment containing Ehcp112 and Ehadh genes, which encode for the proteins of the EhCPADH complex, involved in phagocytosis. This particular genetic organisation suggests that the three corresponding proteins may be functionally related. Here, we studied the relationship of EhRabB with EhCPADH and actin during phagocytosis. First, we obtained the EhRabB 3D structure to carry out docking analysis to predict the interaction sites involved in the EhRabB protein and the EhCPADH complex contact. By confocal microscopy, transmission electron microscopy, and immunoprecipitation assays, we revealed the interaction among these proteins when they move through different vesicles formed during phagocytosis. The role of the actin cytoskeleton in this event was also confirmed using Latrunculin A to interfere with actin polymerisation. This affected the movement of EhRabB and EhCPADH, as well as the rate of phagocytosis. Mutant trophozoites, silenced in EhrabB gene, evidenced the interaction of this molecule with EhCPADH and strengthened the role of actin during erythrophagocytosis.

Identification of the gene encoding the TATA box-binding protein-associated factor 1 (TAF1) and its putative role in the heat shock response in the protozoan parasite Entamoeba histolytica.

Transcription factor IID (TFIID) is a cornerstone in the transcription initiation in eukaryotes. It is composed of TBP and approximately 14 different subunits named TBP-associated factors (TAFs). TFIID has a key role in transcription of many genes involved in cell proliferation, cell growth, cell cycle, cell cycle checkpoint, and various other processes as well. Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, represents a major global health concern. Our research group has previously reported the genes coding the TATA box-binding protein (EhTBP) and TBP-related factor 1 (EhTRF1), which displayed different mRNA levels in trophozoites under different stress conditions. In this work, we identified the TBP-associated factor 1 (Ehtaf1) gene in the E. histolytica genome, which possess a well-conserved DUF domain and a Bromo domain located in the middle and C-terminus of the protein, respectively. The EhTAF1-DUF domain tertiary structure is similar to the corresponding HsTAF1 DUF domain. RT-qPCR experiments with RNA isolated from trophozoites harvested at different time points of the growth curve and under different stress conditions revealed that the Ehtaf1 gene was found slightly upregulated in the death phase of growth curve, but under heat shock stress, it was found upregulated 10 times, suggesting that Ehtaf1 might have an important role in the heat shock stress response. We also found that EhTAF1 is expressed in the nucleus and cytoplasm at 37 °C, but under heat shock stress, it is overexpressed in both the nucleus and cytoplasm, and partially colocalized with EhHSP70 in cytoplasm.

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.

Telomeric Repeat-Binding Factor Homologs in Entamoeba histolytica: New Clues for Telomeric Research.

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

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.

The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair.

The protozoan parasite Entamoeba histolytica is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. E. histolytica harbors enzymes involved in DNA repair pathways like Base and Nucleotide Excision Repair. The majority of DNA repairs pathways converge in their final step in which a DNA ligase seals the DNA nicks. In contrast to other eukaryotes, the genome of E. histolytica encodes only one DNA ligase (EhDNAligI), suggesting that this ligase is involved in both DNA replication and DNA repair. Therefore, the aim of this work was to characterize EhDNAligI, its ligation fidelity and its ability to ligate opposite DNA mismatches and oxidative DNA lesions, and to study its expression changes and localization during and after recovery from UV and H2O2 treatment. We found that EhDNAligI is a high-fidelity DNA ligase on canonical substrates and is able to discriminate erroneous base-pairing opposite DNA lesions. EhDNAligI expression decreases after DNA damage induced by UV and H2O2 treatments, but it was upregulated during recovery time. Upon oxidative DNA damage, EhDNAligI relocates into the nucleus where it co-localizes with EhPCNA and the 8-oxoG adduct. The appearance and disappearance of 8-oxoG during and after both treatments suggest that DNA damaged was efficiently repaired because the mainly NER and BER components are expressed in this parasite and some of them were modulated after DNA insults. All these data disclose the relevance of EhDNAligI as a specialized and unique ligase in E. histolytica that may be involved in DNA repair of the 8-oxoG lesions.

The Conserved ESCRT-III Machinery Participates in the Phagocytosis of Entamoeba histolytica.

The endosomal sorting complex required for transport (ESCRT) orchestrates cell membrane-remodeling mechanisms in eukaryotes, including endocytosis. However, ESCRT functions in phagocytosis (ingestion of ≥250 nm particles), has been poorly studied. In macrophages and amoebae, phagocytosis is required for cell nutrition and attack to other microorganisms and cells. In Entamoeba histolytica, the voracious protozoan responsible for human amoebiasis, phagocytosis is a land mark of virulence. Here, we have investigated the role of ESCRT-III in the phagocytosis of E. histolytica, using mutant trophozoites, recombinant proteins (rEhVps20, rEhVps32, rEhVps24, and rEhVps2) and giant unilamellar vesicles (GUVs). Confocal images displayed the four proteins located around the ingested erythrocytes, in erythrocytes-containing phagosomes and in multivesicular bodies. EhVps32 and EhVps2 proteins co-localized at the phagocytic cups. Protein association increased during phagocytosis. Immunoprecipitation and flow cytometry assays substantiated these associations. GUVs revealed that the protein assembly sequence is essential to form intraluminal vesicles (ILVs). First, the active rEhVps20 bound to membranes and recruited rEhVps32, promoting membrane invaginations. rEhVps24 allowed the detachment of nascent vesicles, forming ILVs; and rEhVps2 modulated their size. The knock down of Ehvps20 and Ehvps24genes diminished the rate of erythrophagocytosis demonstrating the importance of ESCRT-III in this event. In conclusion, we present here evidence of the ESCRT-III participation in phagocytosis and delimitate the putative function of proteins, according to the in vitro reconstruction of their assembling.