Abstract

Enteric amoebae of the genus Entamoeba travel from host to host in an encysted form. We previously showed that in vitro cyst development of Entamoeba invadens requires the addition of defined amounts of multivalent galactose-terminated molecules, such as mucin, to the cultures. The amoeba surface lectin that binds mucin is presumed to convey transmembrane signals when clustered by the ligand, but the signaling molecules that function downstream of the lectin are not known. We report here that Entamoeba encystation was induced in the absence of galactose ligand when catecholamines were added to the encystation medium. Micromolar amounts of both epinephrine and norepinephrine induced encystation. Of a variety of synthetic catecholamine agonists tested, only beta(1)-adrenergic receptor agonists supported encystation, whereas alpha- and beta(2)-adrenergic receptor agonists did not. Only beta(1)-adrenergic receptor antagonists inhibited encystation, and did so even when exogenous catecholamines were not added, indicating that catecholamine binding is required for encystation and suggesting an endogenous source of the ligand. High performance liquid chromatography analysis of Entamoeba extracts showed that the amoebae themselves contain catecholamines and at least one of these is released when the cells are stimulated to encyst with galactose-terminated ligands. The presence of catecholamine binding sites on the surface of amoeba trophozoites was confirmed using radiolabeled catecholamine antagonist. Amoeba encystment was inhibited by addition of beta(1)-adrenergic receptor antagonist to cells that were stimulated to differentiate with either galactose ligand or catecholamines, but not with dibutyryl cAMP. This suggests that the amoeba catecholamine receptor functions downstream of the galactose lectin and upstream of adenylyl cyclase. This enteric protozoan parasite, therefore, contains the components of an autocrine catecholamine ligand-receptor system that may act in conjunction with a galactose lectin to regulate differentiation into the infectious cyst stage.

Highlights

  • Multiplies within the colon of the host, and a dormant cyst stage that is passed into the external environment to infect another host

  • We report here that Entamoeba encystation was induced in the absence of galactose ligand when catecholamines were added to the encystation medium

  • Exogenous Catecholamines Stimulate Encystation of Entamoebae—We had previously found that efficient (Ն90%) in vitro encystment of E. invadens required the presence in the cyst induction medium of ligands for a cell surface galactose lectin (9), and that much lower levels (10 –25%) of cyst formation occurred in their absence

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Summary

EXPERIMENTAL PROCEDURES

Materials—[3H](Ϫ)-CGP 12177 was obtained from PerkinElmer Life Sciences. Waters AccQ-Fluor reagent kit was obtained from Millipore (Milford, MA). For analysis of medium from high and low cell density cultures, E. invadens trophozoites were harvested from 50-ml cultures in BYI-S-33 and washed with ice-cold AWB. For analysis of encystation medium, the amoebae were resuspended to yield a final concentration of 5 ϫ 105 trophozoites/ml in encystation medium containing 0.1 ng/ml, 1 ␮g/ml, or 100 ␮g/ml ASF in 5-ml glass screw-cap tubes. E. invadens and E. histolytica trophozoites were harvested from cultures grown in BYI-S-33 as described above and induced to encyst in 100% LG encystation medium containing 1 ␮g/ml ASF or 5% ABS for 15 min at room temperature. E. invadens tropohozoites were harvested from cultures grown in BYI-S-33 as described above, and induced to encyst in 47% LG medium containing 1 ␮g/ml ASF for 15 min at room temperature. The cells were processed over glass fiber filters as above

RESULTS
Percentage of encystation
Binding conditions
DISCUSSION
TABLE III Catecholamine content of Entamoeba trophozoites

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