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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.

Acanthamoeba spp. is the etiological agent of amoebic keratitis. In this study, the effect of taurine in physiological concentrations in tears (195 μM) on trophozoites of Acanthamoeba castellanii through the ex vivo amoebic keratitis model was evaluated. Trophozoites were coincubated with the Syrian golden hamster cornea (Mesocricetus auratus) for 3 and 6 h. Group 1: Control (−). Corneas coincubated with amoebic culture medium and taurine. Group 2: Control (+). Corneas coincubated with trophozoites without taurine. Group 3: Corneas coincubated with taurine 15 min before adding trophozoites. Group 4: Trophozoites coincubated 15 min with taurine before placing them on the cornea. Group 5: Corneas coincubated for 15 min with trophozoites; subsequently, taurine was added. Results are similar for both times, as evaluated by scanning electron microscopy. As expected, in the corneas of Group 1, no alterations were observed in the corneal epithelium. In the corneas of Group 2, few adhered trophozoites were observed on the corneal surface initiating migrations through cell junctions as previously described; however, in corneas of Groups 3, 4 and 5, abundant trophozoites were observed, penetrating through different corneal cell areas, emitting food cups and destabilizing corneal surface in areas far from cell junctions. Significant differences were confirmed in trophozoites adherence coincubated with taurine (p < 0.05). Taurine does not prevent the adhesion and invasion of the amoebae, nor does it favor its detachment once these have adhered to the cornea, suggesting that taurine in the physiological concentrations found in tears stimulates pathogenic mechanisms of A. castellanii.

The leader of the capsid (LC) protein is exclusive to the Vesivirus genus, and it is needed for successful feline calicivirus (FCV) replication, as well as an efficient apoptosis induction through the mitochondrial pathway. In this work, we aimed to determine if the LC protein from the FCV is a viroporin. Although lacking in a transmembrane domain or an amphipathic helix, the LC protein from the FCV is toxic when expressed in bacteria and it oligomerizes through disulfide bonds, which are both key characteristics of viroporins. An electron microscopy analysis of LC-expressing E. coli cells suggest that the protein induces osmotic stress. Moreover, we found that the previously studied C40A LC mutant, that fails to induce apoptosis and that hinders the replication cycle, also oligomerizes but it has a reduced toxicity and fails to induce osmotic stress in bacteria. We propose that the LC protein is a viroporin that acts as a disulfide bond-dependent antimicrobial peptide, similar to the Ebola virus delta peptide.

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.

Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation.