Compounds of the upper gastrointestinal tract induce rapid and efficient excystation of Entamoeba invadens

Abstract

The infective stage of Entamoeba parasites is an encysted form. This stage can be readily generated in vitro, which has allowed identification of stimuli that trigger the differentiation of the parasite trophozoite stage into the cyst stage. Studies of the second differentiation event, emergence of the parasite from the cyst upon infection of a host, have been hampered by the lack of an efficient means to excyst the parasite and complete the life cycle in vitro. We have determined that a combination of exposures to water, bicarbonate and bile induces rapid excystment of Entamoeba invadens cysts. The high efficiency of this method has allowed the visualization of the dynamics of the process by electron and confocal microscopy, and should permit the analysis of stage-specific gene expression and high through-put screening of inhibitory compounds.

Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns

“…For excystation experiments, trophozoites were encysted for 72 h using the above method and any remaining trophozoites were lysed by incubating overnight (~16 h) in distilled water at 4°C (a step that both lyses trophozoites and enhances excystation). The remaining cysts were induced to excyst by incubating in LYI-LG with 1 mg/ml of bile, 40 mM sodium bicarbonate, 1% glucose and 10% serum for 2 h or 8 h as previously described (Mitra et al, 2010). …”

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

“…For excystation, the encysted parasites were incubated overnight in distilled water at 4°C to lyse the trophozoites. The parasites were then excysted by the addition of bile salts and sodium bicarbonate in LYI-LG for 8 h (44).…”

Ethanol and Isopropanol in Concentrations Present in Hand Sanitizers Sharply Reduce Excystation of Giardia and Entamoeba and Eliminate Oral Infectivity of Giardia Cysts in Gerbils

“…E. invadens cysts were treated with alcohols, and then DAPI and PI labeling of the cysts were performed as described for Giardia, except cyst walls were labeled with 10 g/ml WGA, which binds to chitin fibrils (21). E. invadens excystation rates were determined by incubating 10 4 cysts in 10 ml of excystation medium containing 40 mM sodium bicarbonate and 1.25 mM taurodeoxycholate for 24 h at room temperature and counting intact versus excysted walls with the phase microscope (24). Alternatively, ϳ10…”

“…Cyst wall glycoproteins also contain unique O-phosphodiester-linked glycans (22). When cysts of Giardia or Entamoeba are placed in excystation media that mirror conditions in the small intestines, trophozoites (the motile, colonizing form) are released (23,24).…”

“…Cyst wall glycoproteins also contain unique O-phosphodiester-linked glycans (22). When cysts of Giardia or Entamoeba are placed in excystation media that mirror conditions in the small intestines, trophozoites (the motile, colonizing form) are released (23,24).We are interested in alcohol-based hand sanitation methods to reduce transmission of Giardia and Entamoeba, because these sanitizers, which were identified Ͼ100 years ago, are effective against many viral, bacterial, and fungal infections; alcohol-based sanitizers are relatively inexpensive and do not depend upon scarce supplies of clean water; and there are no human vaccines for Giardia and Entamoeba (25-32). Hand sanitizers contain 63% to 80% ethanol or isopropanol.…”