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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.

Naegleria fowleri causes a fatal disease known as primary amoebic meningoencephalitis. This condition is characterized by an acute inflammation that originates from the free passage of peripheral blood cells to the central nervous system through the alteration of the blood-brain barrier. In this work, we established models of the infection in rats and in a primary culture of endothelial cells from rat brains with the aim of evaluating the activation and the alterations of these cells by N. fowleri. We proved that the rat develops the infection similar to the mouse model. We also found that amoebic cysteine proteases produced by the trophozoites and the conditioned medium induced cytopathic effect in the endothelial cells. In addition, N. fowleri can decrease the transendothelial electrical resistance by triggering the destabilization of the tight junction proteins claudin-5, occludin, and ZO-1 in a time-dependent manner. Furthermore, N. fowleri induced the expression of VCAM-1 and ICAM-1 and the production of IL-8, IL-1β, TNF-α, and IL-6 as well as nitric oxide. We conclude that N. fowleri damaged the blood-brain barrier model by disrupting the intercellular junctions and induced the presence of inflammatory mediators by allowing the access of inflammatory cells to the olfactory bulbs.

Summary Lysine methylation of histones, a posttranslational modification catalyzed by lysine methyltransferases (HKMTs), plays an important role in the epigenetic regulation of transcription. Lysine methylation of non-histone proteins also impacts the biological function of proteins. Previously it has been shown that lysine methylation of histones of Entamoeba histolytica, the protozoan parasite that infects 50 million people worldwide each year and causing up to 100,000 deaths annually, is implicated in the epigenetic machinery of this microorganism. However, the identification and characterization of HKMTs in this parasite had not yet been determined. In this work we identified four HKMTs in E. histolytica (EhHKMT1 to EhHKMT4) that are expressed by trophozoites. Enzymatic assays indicated that all of them are able to transfer methyl groups to commercial histones. EhHKMT1, EhHKMT2 and EhHKMT4 were detected in nucleus and cytoplasm of trophozoites. In addition EhHKMT2 and EhHKMT4 were located in vesicles containing ingested cells during phagocytosis, and they co-immunoprecipitated with EhADH, a protein involved in the phagocytosis of this parasite. Results suggest that E. histolytica uses its HKMTs to regulate transcription by epigenetic mechanisms, and at least two of them could also be implicated in methylation of proteins that participate in phagocytosis.

Background information. JAM-C (junctional adhesion molecule C) has been implicated in the regulation of leukocyte migration, cell polarity, spermatogenesis, angiogenesis and nerve conduction. JAM-C has been also reported to concentrate at TJs (tight junctions) and desmosomes, although detailed localization studies remain incomplete. Results. Monoclonal (LUCA14, MAB1189, Gi11, and PACA4) and polyclonal (40-9000) antibodies were employed to evaluate JAM-C expression/localization in various epithelial cell lines. However, RT—PCR (reverse transcription—PCR) assays revealed no JAM-C mRNA in SK-CO15, HeLa and HPAF-II cells, whereas abundant mRNA was detected in platelets, Caco-2 and ARPE cells. Interestingly, immunofluorescence localization in all cells revealed strong intercellular junctional staining with all of the above antibodies, except PACA4. Given the positive staining results in cells lacking JAM-C mRNA, immunoblot analyses were performed. Western blots revealed a prominent protein band at 52 kDa in all cells tested with all antibodies except PACA4. However, the correct size of JAM-C (37 kDa) was only detected in cells containing JAM-C mRNA. Immunofluorescence staining of JAM-C mRNA-expressing Caco-2 cells using mAb PACA4 revealed co-localization with occludin and ZO-1 (zonula occludens 1) at TJs. Analyses by MS identified the cross-reactive 52 kDa protein band as K8 (keratin 8). Furthermore, siRNA (small interfering RNA)-mediated downregulation of K8 in JAM-C mRNA-negative cells resulted in diminished junctional staining along with a reduction in the intensity of the 52 kDa protein band. Using an antibody specific for K8 phosphorylated at Ser73, the 52 kDa protein was identified as this phosphorylated form of K8. Conclusions. The results from the present study demonstrate that a majority of available anti-human JAM-C antibodies cross-react with phosphorylated K8 and suggest that cellular localization studies using these reagents should be interpreted with caution. Of the JAM-C antibodies tested, only mAb PACA4 is monospecific for human JAM-C. Analyses using PACA4 reveal that JAM-C expression is variable in different epithelial cell lines with co-localization at TJs.

A stable intestinal epithelium is the basis for healthy intestines. Its dysfunction causes increased intestinal permeability and bacterial translocation that may eventually cause an aberrant immune...

Entamoeba histolytica is the causative agent of human amoebiasis. 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). We recently showed that the E. histolytica complex EhCPADH112, formed by the EhADH112 adhesin and the EhCP112 cysteine protease, participates during epithelial invasion. To elucidate the particular role of EhADH112 in this process, we overexpressed this adhesion molecule in MDCK epithelial cells. Expression of EhADH112 was proven by RT-PCR and western blot assays. EhADH112 localized at the plasma membrane of epithelial cells, similar to its location in trophozoites. EhADH112 improved adhesion between epithelial cells induced by an increase in TER and expression of TJ proteins. Importantly, infection of MDCK cells expressing EhADH112 with trophozoites caused a faster drop in TER and more epithelial damage compared to control cells; suggesting that EHADH112 in ep...