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Entamoeba histolytica , the causative agent of amoebiasis, does not form cysts in vitro , so reptilian pathogen Entamoeba invadens is used as an Entamoeba encystation model. During the in vitro encystation of E. invadens , a few multinucleated giant cells (MGC) were also appeared in the culture along with cysts. Like the cyst, these MGC's were also formed in the multicellular aggregates found in the encystation culture. Time-lapse live cell imaging revealed that MGC's were the result of repeated cellular fusion with fusion-competent trophozoites as a starting point. The early MGC were non-adherent, and they moved slowly and randomly in the media, but under confinement, MGC became highly motile and directionally persistent. The increased motility resulted in rapid cytoplasmic fissions, which indicated the possibility of continuous cell fusion and division taking place inside the compact multicellular aggregates. Following cell fusion, each nucleus obtained from the fusion-competent trophozoites gave rise to four nuclei with half genomic content. All the haploid nuclei in MGC later aggregated and fused to form a polyploid nucleus. These observations have important implications on Entamoeba biology as they point toward the possibility of E. invadens undergoing sexual or parasexual reproduction.

Amoebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. This parasite undergoes a fundamental cell differentiation process from proliferative trophozoite to dormant cyst, termed "encystation." The cysts formed by encystation are solely responsible for the transmission of amoebiasis; therefore, Entamoeba encystation is an important subject from both biological and medical perspectives. Here, we have established a flow cytometry strategy for not only determining the percentage of formed cysts but also for monitoring changes in cell populations during encystation. This strategy together with fluorescence microscopy enables visualization of the cell differentiation process of Entamoeba encystation. We also standardized another flow cytometry protocol for counting live trophozoites. These two different flow cytometry techniques could be integrated into 96-well plate-based bioassays for monitoring the processes of cyst formation and trophozoite proliferation, which are crucial to maintain the Entamoeba life cycle. The combined two systems enabled us to screen a chemical library, the Pathogen Box of the Medicine for Malaria Venture, to obtain compounds that inhibit either the formation of cysts or the proliferation of trophozoites, or both. This is a prerequisite for the development of new drugs against amoebiasis, a global public health problem. Collectively, the two different 96-well plate-based Entamoeba bioassay and flow cytometry analysis systems (cyst formation and trophozoite proliferation) provide a methodology that can not only overcome the limitations of standard microscopic counting but also is effective in applied as well as basic Entamoeba biology.

Entamoeba histolytica causes dysentery and liver abscess mostly in countries that lack proper sanitation. Infection is acquired by ingestion of the cyst form in contaminated food or water. E. histolytica does not encyst in vitro; thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a surrogate. Cysts are small and possess chitin-rich walls. These are characteristics that may be exploited by flow cytometry. We stained encysting E. invadens cells with a fluorescent chitin stain, and analyzed fluorescence and forward scatter by flow cytometry. We demonstrate that flow cytometry can be used to track differentiation, reveal unique cell populations, and evaluate encystation inhibitors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D. These data demonstrate different structures and expressions of four chitinases in the differentiation of E. invadens.