Acid Intracellular Vesicles and the Cytolysis of Mammalian Target Cells by Entamoeba histolytica Trophozoites1

Abstract

Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HMI strain of E. histolytica and measure vesicle pH (5.1 .+-. 0.2 by spectrofluorimetry). Concentrations of NH4Cl (1.0-10.0 mM) sufficient to increase vesicle pH to .gtoreq. 5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of 3H-thymidine previously incorporated into CHO cell monolayers, and by the release of 111indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 .mu.M). In contrast, NH4Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4.degree.C and by the binding and ingestion of 3H-leucine-labeled bacteria. In the presence of NH4Cl and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 .mu.g/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH4Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 .mu.g for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.