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-throughput screening of inhibitory compounds.

Fig. 1

Effect of various individual treatments on the excystation of in vitro generated Entamoeba invadens cysts. (A) 8×10⁵ detergent-resistant cysts pre-treated with distilled water for the indicated times were placed in TYI-S-33-based media (TYI) and excystment was examined at 24 and 48 h. To optimize the concentration of sodium bicarbonate (B), taurocholate (C), taurodeoxycholate (D) and bile (E), detergent-resistant cysts were transferred directly to TYI containing the indicated concentrations of each substance, and excystment was examined at 24 and 48 h. Error bars represent the S.E.M. of three independent experiments. C, control.

Fig. 2

Combined effects of water pre-treatment, sodium bicarbonate (SBC) and bile/bile salts on Entamoba invadens excystation. (A) Approximately 8×10⁵ cysts were incubated in TYI-S-33-based media after pre-treatment of cysts in water for 6 h, with or without subsequent addition of SBC, bile/bile salts or a combination of the two. The excystation percentage was determined after 24 h. (B) Kinetics of excystation. Cysts were incubated as in (A) and at the indicated times, cysts and trophozoites were harvested and the excystation percentage was determined.

Fig. 3

Numbers of nuclei per cell before and during excystation of Entamoeba invadens. Seventy-two h detergent-resistant cysts were excysted for the indicated times (B–F), collected by centrifugation, stained with Hoechst dye, and the numbers of nuclei per cell were determined using a Nikon Eclipse E600 fluorescence microscope. A) A photograph of the stained cysts corresponding to B. Error bars represent the S.E.M.

Fig. 4

Effect of sodium bicarbonate and bile on growth of Entamoeba invadens trophozoites. Trophozoites (1.5×10⁴ cells/ml) were placed in TYI-S-33-based media with sodium bicarbonate (40 mM), or bile (1 mg/ml) or both. At 24 h intervals cells were counted. Error bars represent the S.E.M. of two independent experiments.

Fig. 5

Visual quantification of excystment of Entamoeba invadens. (A) Light micrographs of semi-thin Epon sections. After 6 h water pre-treatment, cysts were incubated in TYI-S-33-based media in the presence of sodium bicarbonate and bile acid. Samples were collected at 0, 2, 4, 6 and 8 h time points, fixed and embedded, and semi-thin sections were stained and examined by light microscopy. Scale bar = 10 μm. (B) Quantitative analysis of excysted trophozoites and intact cysts of semi-thin sections as shown in A. Error bars represent the S.E.M.

Fig. 6

Sequential events of Entamoeba invadens excystation. Transmission electron micrographs of typical ultrastructural features of cysts and excysting cells during the first 8 h following excystment induction. A and B) Cysts prior to excystment, containing chromatoid bodies (CB) and multiple nuclei (N). Note that the plasma membrane is closely apposed to the inner aspect of the cyst wall. As excystment progresses, spaces (arrows) between the parasite and the cyst wall develop (C–G). D and E) Cells with an invagination of the trophozoite located between flanking points of contact with the cyst wall (arrow heads). Depending on the plane of section, the rest of the trophozoite appears to be completely separated from the cyst wall (F) or attached in a very limited way (G). H) A trophozoite that appears to be exiting from the cyst capsule. I and J) Excysted trophozoites.

Fig. 6

Sequential events of Entamoeba invadens excystation. Transmission electron micrographs of typical ultrastructural features of cysts and excysting cells during the first 8 h following excystment induction. A and B) Cysts prior to excystment, containing chromatoid bodies (CB) and multiple nuclei (N). Note that the plasma membrane is closely apposed to the inner aspect of the cyst wall. As excystment progresses, spaces (arrows) between the parasite and the cyst wall develop (C–G). D and E) Cells with an invagination of the trophozoite located between flanking points of contact with the cyst wall (arrow heads). Depending on the plane of section, the rest of the trophozoite appears to be completely separated from the cyst wall (F) or attached in a very limited way (G). H) A trophozoite that appears to be exiting from the cyst capsule. I and J) Excysted trophozoites.

Fig. 7

Live cell imaging of excysting Entamoeba invadens trophozoites. Cysts were labeled with calcofluor (cyst wall, blue) and SYTO11 (nuclei, green) following exposure to the water/bile/bicarbonate excystment medium, and viewed by confocal microscopy. Differential interference contrast was used to visualize the outline of the cells. A) A typical excystment sequence, with the arrow indicating the cyst wall of the excysting cell. At 244 s, all four nuclei of the emerging trophozoite are visible in the optical plane. Numbers represent seconds elapsed after the first frame, and the scale bar represents 20 μm. B) Discarded cyst walls from trophozoites that have already excysted. Scale bar represents 15 μm.