cytochalasin-b and Amebiasis

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process.

Topics: Acanthamoeba castellanii; Acetylglucosamine; Actin Cytoskeleton; Amebiasis; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cell Line; Cell Surface Extensions; Cytochalasin B; Cytoskeleton; Epithelial Cells; Glycoproteins; Host-Parasite Interactions; Humans; Lectins; Mannose; Models, Biological; Protozoan Proteins; Thiazolidines; Trophozoites

Cinemicrography of Entamoeba histolytica destruction of Chinese hamster ovary (CHO) cells shows that ameba cytopathogenicity consists of separate components: a contact-dependent cytolethal effect, and phagocytosis. Cells not in contact with amebae remain intact. Quantitation of ameba destruction of CHO cells by applying the one-hit hypothesis confirms that the cytoethal effect of amebae is contact dependent. Studies with 111Indium oxine-labeled cells provide further evidence of extracellular killing by E. histolytica and indicate that > 94% of the target cells are killed before phagocytosis. When we examined for a cytotoxin release by E. histolytica, we found no effect on CHO cells with filtrates of amebae, and a nonspecific effect of cell rounding and release with sonicates of amebae. The ameba sonicate effect was time-dose dependent, was not cytolethal, was reversible, and was inhibited by alpha II macroglobulin. Cytochalasin B altered ameba motility and morphology, and monolayer experiments confirmed that cytochalasins A, B, or D inhibited CHO cell destruction by E. histolytica. Cytochalasin D also inhibited extracellular killing of CHO cells by amebae in pellets, apparently independent of effects on ameba motility or phagocytosis. Colchicine and vinblastine, alone or in combination with cytochalasin D, did not inhibit E. histolytica cytopathogenicity, which indicates that microtubule function is not required for target cell killing by amebae.

Topics: Amebiasis; Animals; Cells, Cultured; Colchicine; Cricetinae; Cricetulus; Cytochalasin B; Cytoskeleton; Entamoeba histolytica; Entamoebiasis; Female; Microtubules; Ovary; Time Factors; Vinblastine; Virulence