STUDIES ON THE ENCYSTATION OF ENTAMOEBA

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Using an axenic encystation system of Entamoeba invadens as a model for E. histolytica encystation, effect of aphidicolin, a specific inhibitor of nuclear replicative DNA polymerases, on the encystation was examined. Aphidicolin inhibited the encystation and its effect was reversible. Encystation was almost completely inhibited when trophozoites were pretreated with the drug. This can be explained that treatment with aphidicolin accumulates the cells at G1/S border and those cells can neither synthesize DNA nor divide when placed in encystation medium, whereas the cells without the pretreatment include G2 and M cells which have already synthesized DNA so that they are not affected by aphidicolin. The level of DNA polymerase activity in cysts decreased as encystation proceeded as compared with that of trophozoites. The results indicate that encystation is accompanied by a reduced level of DNA polymerase activity, which correlates with the previous finding that nuclear division occurs du … More ring cyst maturation in the absence of DNA synthesis. The appearance of cyst-specific proteins in encysting E. invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. A rabbit antiserum against cysts reacted with the trophozoite proteins as well as cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific and the protein was found to be present in the particulate fraction, which was rich in cell-wall fragments. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall. Effect of cytochalasin D, a specific inhibitor of microfilaments, on the encystation of E. invadens was examined. Encystation was inhibited by cytochalasin D in a dose-dependent manner. This is the first evidence for participation of microfilaments in the encystation. Trophozoites grown with cytochalasin D became multinucleate and produced multinucleate cysts when transferred to encystation medium. Encystation with cytochalasin D of trophozoites grown with the drug was more inhibited than that of trophozoites grown without the drug. Thus multinucleation of trophozoites by cytochalasin D had an inhibitory effect on the encystation. Less