A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

Abstract

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.

The dynamics of ultrastructural changes during Entamoeba invadens encystation

“…We revealed the dynamics of ultrastructure changes during E. invadens encystation. Furthermore, we combined a flow cytometry method to monitor the level of cell differentiation in a population of live cells (Mi-Ichi et al, 2018). Our approach enabled us to refine the timing for cyst wall formation because the electron microscopy and flow cytometry monitored cyst wall formation from different perspectives.…”

“…For flow cytometry, cells pelleted in 96-well plates were treated and processed, and the obtained data were analysed as previously described (Mi-Ichi et al, 2018). Excystation was induced as described (Mitra et al, 2010) with slight modification.…”

Repurposing of an old drug: In vitro and in vivo efficacies of buparvaquone against Echinococcus multilocularis

“…So far, the Pathogen Box has been screened against the nematode H. contortus ( Preston et al, 2016 ), the fungi Candida albicans and Cryptococcus neoformans ( Vila and Lopez-Ribot, 2016 ; Mayer and Kronstad, 2017 ), Plasmodium and the kinetoplastids Leishmania and Trypanosoma ( Calit et al, 2018 ; Dennis et al, 2018 ; Duffy et al, 2017 ), Neospora caninum ( Müller et al, 2017 ) , Mycobacterium abscessus and M. avium ( Jeong et al, 2018 ; Low et al, 2017 ), Toxoplasma gondii ( Spalenka et al, 2017 ), C. elegans ( Partridge et al, 2018 ), Entamoeba histolytica ( Mi-Ichi et al, 2018 ), and Giardia lamblia and Cryptosporidium parvum ( Hennessey et al, 2018 ). Interestingly, all compounds that exhibited activity against E. multilocularis were also active against at least one more pathogen other than the one it was selected for by MMV (with the exception of nitazoxanide), thus underlining the importance and potential of the concept of drug repurposing.…”

Characterization of Entamoeba histolytica adenosine 5′-phosphosulfate (APS) kinase; validation as a target and provision of leads for the development of new drugs against amoebiasis

“…The storage conditions of the 400 compounds in the Pathogen Box, obtained from MMV, and the supplier and storage condition of auranofin (>98% purity) (which is E-H-05 in the Pathogen Box) were described in [18]. 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide (>96% purity) and 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide (>96% purity), which are A-D-11 and A-H-11, respectively, in the Pathogen Box, were purchased from Enamine Ltd. (Kiev, Ukraine), dissolved in dimethyl sulfoxide (DMSO) at 10 mM as stock solutions and stored at -30°C in 40 μL aliquots.…”

“…As well as appropriate targets, providing desirable leads is essential for the development of new drugs; therefore, a variety of methods for screening large numbers of compounds have been undertaken as the prerequisite step. For instance, high through-put in vitro assays using recombinant enzymes [16, 17], wild-type cells [18–20], or an engineered cell [20] have been used. However, screening a huge number of compounds, e .…”