Influence of Micropatterned Grill Lines on Entamoeba histolytica Trophozoites Morphology and Migration

Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a–better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified

Surface Coat Differences between Invasive Entamoeba histolytica and Non-Invasive Entamoeba dispar

Using ultrastructural cytochemical techniques we have found differences in the distribution of surface coat components between the invasive protozoan parasite Entamoeba histolytica and the non-invasive Entamoeba dispar. Carbohydrate-containing components and anionic sites in the cell surface of both species were detected by staining with ruthenium red and cationized ferritin, respectively. Ruthenium red staining revealed a thicker surface coat in E. histolytica trophozoites, whereas trophozoites of E. dispar showed a higher concentration of cationized ferritin particles on its surface. Mannose or glucose residues were found at the plasma membrane of both parasites treated with Concanavalin A (Con A)-peroxidase; the surface reaction product was more evident in E. dispar, compared with E. histolytica. Con A rapidly produced surface caps in E. histolytica trophozoites, whereas E. dispar showed a much less efficient mobilization of surface Con A receptors. Agglutination with Con A produced much larger clumps in E. histolytica in comparison with E. dispar. In turn, biotinylation assays revealed striking differences in the composition of surface membrane proteins in both amebic species. Overall, these results further emphasize the phenotypic differences between these two common parasites of the human intestinal tract, once considered to be the same protozoan

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

Entamoeba histolytica Up-Regulates MicroRNA-643 to Promote Apoptosis by Targeting XIAP in Human Epithelial Colon Cells

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. Recent evidences suggested that host cells miRNAs are involved in the progression of infectious diseases, but its role in amoebiasis remains largely unknown. Here, we reported an unexplored role for miRNAs of human epithelial colon cells during the apoptosis induced by Entamoeba histolytica. We demonstrated for the first time that SW-480 colon cells change their miRNAs profile in response to parasite exposure. Our data showed that virulent E. histolytica trophozoites induced apoptosis of SW-480 colon cells after 45 min interaction, which was associated to caspases-3 and -9 activation. Comprehensive profiling of 667 miRNAs using Taqman Low-Density Arrays showed that 6 and 15 miRNAs were significantly (FC > 1.5; p < 0.05) modulated in SW-480 cells after 45 and 75 min interaction with parasites, respectively. Remarkably, no significant regulation of the 6-miRNAs signature (miR-526b-5p, miR-150, miR-643, miR-615-5p, miR-525, and miR-409-3p) was found when SW-480 cells were exposed to non-virulent Entamoeba dispar. Moreover, we confirmed that miR-150, miR-643, miR-615-5p, and miR-525 exhibited similar regulation in SW-480 and Caco2 colon cells after 45 min interaction with trophozoites. Exhaustive bioinformatic analysis of the six-miRNAs signature revealed intricate miRNAs-mRNAs co-regulation networks in which the anti-apoptotic XIAP, API5, BCL2, and AKT1 genes were the major targets of the set of six-miRNAs. Of these, we focused in the study of functional relationships between miR-643, upregulated at 45 min interaction, and its predicted target X-linked inhibitor of apoptosis protein (XIAP). Interestingly, interplay of amoeba with SW-480 cells resulted in downregulation of XIAP consistent with apoptosis activation. More importantly, loss of function studies using antagomiRs showed that forced inhibition of miR-643 leads to restoration of XIAP levels and suppression of both apoptosis and caspases-3 and -9 activation. Congruently, mechanistic studies using luciferase reporter assays confirmed that miR-643 exerts a postranscripcional negative regulation of XIAP by targeting its 3′-UTR indicating that it's a downstream effector. In summary, we provide novel lines of evidence suggesting that early-branched eukaryote E. histolytica may promote apoptosis of human colon cells by modulating, in part, the host microRNome which highlight an unexpected role for miRNA-643/XIAP axis in the host cellular response to parasites infection

Evidence for a “Wattle and Daub” Model of the Cyst Wall of Entamoeba

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins)

In vitro Induction of Entamoeba histolytica Cyst-like Structures from Trophozoites

Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment

Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection

Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors

Comportamiento clínico epidemiológico del dengue en pacientes pediátricos ingresados en el Hospital Escuela Oscar Danilo Rosales Argüello de junio - diciembre 2018

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