Flow cytometric characterization of encystation in Entamoeba invadens

Abstract

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.

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

“…This study and that of Welter et al ( 2017 ) for determining the effects of compounds on encystation by quantifying E. invadens cyst formation are essentially the same; therefore, the usefulness of flow cytometry to screen compounds that show a significant effect on Entamoeba encystation was confirmed by two distinct procedures. However, as is evident from the present study, an important issue to be addressed is whether each compound directly affects the encystation process, or has an indirect effect by damaging trophozoites.…”

“…To obtain accurate data, reproducible distinction between trophozoites and cysts is essential. Flow cytometry has been recently reported as a suitable method for this (Welter et al, 2017 ). Here, we attempted to introduce and standardize conditions to achieve these criteria.…”

“…Recently, Welter et al reported using flow cytometry for fixed E. invadens prepared from an encystation-inducing culture with the fluorescent dye, Congo Red. This method can separate the trophozoite and cyst populations and assess the effect of compounds on the dynamics of these two populations (Welter et al, 2017 ). In the present study, as well as a fluorescent dye (CF) that stains chitin (Arroyo-Begovich et al, 1980 ), a dye (EB) that stains membrane (Hed et al, 1983 ) were used and more time points during encystation were then analyzed to monitor the dynamics of population changes.…”

Encystation of Entamoeba histolytica in Axenic Culture

“…Flow cytometry has previously been applied as a method for examining encystation in Entamoeba species [ 46 , 47 ], and we likewise used this to analyze E . histolytica trophozoite and cyst populations.…”

“…Flow cytometry analysis has also been used to demonstrate that encystation is accompanied by a decrease in size as cells convert from trophozoite to cyst form [ 47 ]. We performed flow cytometry analysis of E. histolytica cultures grown at high cell density in TYI basal medium.…”